Antibody to human il-1 beta

ABSTRACT

The present invention relates to anti-IL-1 beta binding members and in particular to monovalent high potency IL-1 beta-binding antibody fragments being highly stable and soluble. Such binding members may be used in the treatment of inflammatory and other diseases as well as in diagnostics. Also provided are related nucleic acids, vectors, cells, and compositions.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 16/118,124, filed on Aug. 30, 2018, which is acontinuation of and claims priority to U.S. patent application Ser. No.15/202,982, filed on Jul. 6, 2016, now U.S. Pat. No. 10,077,302, whichis a divisional of and claims priority to U.S. patent application Ser.No. 14/072,165, filed on Nov. 5, 2013, now U.S. Pat. No. 9,404,930,which claims the benefit of U.S. Provisional Application No. 61/722,532,filed on Nov. 5, 2012. This application claims priority under 35 U.S.C.§ 119 or 365 to EP Application No. 12007503.1, filed Nov. 5, 2012. Theentire teachings of the above applications are incorporated herein byreference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 1449-2TSDVCT2_ST25.txt, 224,536 bytes in size, generatedon Feb. 27, 2020 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

DESCRIPTION

The invention relates to humanized anti-IL-1 beta antibodies, inparticular monovalent, highly potent anti-IL-1 beta antibody fragments.The invention also relates to nucleic acids encoding such antibodies,vectors, host cells containing such sequences, pharmaceutical anddiagnostic compositions comprising the antibodies or nucleic acids, anduses thereof.

BACKGROUND OF THE INVENTION

Interleukin-1 beta (IL-1 beta) is a pro-inflammatory cytokine which isproduced as a precursor by activated macrophages. Upon proteolyticcleavage, signal transduction is initiated by binding of the active formto the IL-1 receptor type I (IL-1R1) which in turn associates with thetransmembrane IL-1 receptor accessory protein (IL-1RAP). The formedcomplex is competent of signal transduction. Being a key mediator in theinflammatory response, the cytokine affects a number of cellularactivities such as cell proliferation, differentiation, and apoptosis.Therefore, IL-1 beta has been considered an important target for avariety of pharmaceuticals.

There is a need in the art for antibodies with high therapeuticpotential against human IL-1 beta. For being therapeutically successful,it is important that such antibody displays desirable biophysical andbiochemical characteristics. For example, since the target IL-1 beta isa highly efficient interleukin that is potent at very low concentrationsand thus needs to be comprehensively blocked, such antibody needs to behighly potent as well as highly stable and soluble.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a monovalent antibody fragmentdirected against IL-1 beta having a potency of lower than 50 picomolar(pM), as determined by the half-maximum inhibitory concentration IC₅₀with regard to inhibiting the biological effect of human IL-1 beta.

Monovalent antibody fragments, whether being humanized or not, havingpotency values in the pM-range are particular and not routinelyobtained. In addition and typically, an antibody loses affinity to itstarget upon humanization when compared to the parent non-human antibody.It is therefore a challenge to humanize an antibody such that theaffinity parameters are close or equal to the parent antibody. This isparticularly true for monovalent antibody fragments which comprise onlyone variable light and heavy chain, and therefore bind to the targetless strongly than bivalent antibodies displaying two light and heavychains.

Moreover, when converting a full-length antibody into a smallerfragment, its potency usually becomes diminished. This is not only dueto the accompanying change of valency (for example, the antibodyfragment might only be monovalent whereas a full-length immunoglobulinis bi- or multivalent) but may also be caused by steric reasons.

A potent antibody is particularly useful since it allows administeringlower amounts of drug to the patient, thereby decreasing the overallcosts of treatment. In addition, a more complete neutralization of themolecular target of the disease is rendered feasible.

Moreover, different application routes in animal models as well as inhuman therapy can be envisioned when applying highest potencyantibodies. For example, as to topical drugs, although delivery may belimited due to the barrier function of the epithelial layer, efficacy oftreatment is restored by the high potency of the limited quantity ofdrug molecules that passes this physiological barrier.

Often, the high amount of a less potent drug, which needs to beadministered to achieve similar pharmacodynamic effects, translates intomuch higher intravenous or subcutaneous application volumes than with amore potent drug. Such higher application volumes are a disadvantage foruse in animals and humans for two reasons: firstly, the impracticalityof treating patients with a high volume of drug, and secondly, becauseantibodies are very expensive per unit of mass.

Therefore, lower quantities of antibody used for treatment translateinto lower production costs of the drug. In particular, antibodyfragments are suitable for production using, e.g., bacterial or yeastculture systems, which are of comparatively lower cost than mammalianexpression systems typically used for the production of full-lengthimmunoglobulins such as IgG. The combination of smaller quantities ofdrug to be administered and cheaper manufacturing processes opens thepossibility of more cost-efficient medicines per patient. Thus, a largernumber of patients may benefit from such drug.

Stability and solubility parameters are other factors crucial forproviding a viable medicament. The more stable and soluble an antibodydrug, the smaller the volume of administration and the longer the shelfhalf-life time. The antibodies provided herein are highly stable andsoluble, i.e., they remain monomeric for prolonged periods of time andalso at high concentrations.

In one aspect, an antibody is provided, in particular the monovalentantibody fragment above, comprising:

(a) at least one of the variable heavy chain (VH) complementaritydetermining region (CDR) sequences CDR-H1, CDR-H2 or CDR-H3 as set forthin SEQ ID Nos.: 1, 2 and 3, respectively, or variants thereof; and/or

(b) at least one of the variable light chain (VL) CDR sequences CDR-L1,CDR-L2 or CDR-L3 as set forth in SEQ ID Nos.: 4, 5, and 6, respectively,or variants thereof.

In another embodiment, the antibody, and in particular said monovalentantibody fragment, comprises:

(a) at least one of the variable heavy chain (VH) complementaritydetermining region (CDR) sequences CDR-H1, CDR-H2 or CDR-H3 as set forthin SEQ ID Nos.: 155, 156 and 157, respectively, or variants thereof;

and/or

(b) at least one of the variable light chain (VL) CDR sequences CDR-L1,CDR-L2 or CDR-L3

(i) as set forth in SEQ ID Nos.: 158, 159 and 160, respectively, orvariants thereof, or

(ii) as set forth in SEQ ID Nos.: 161, 162 and 163, respectively, orvariants thereof.

In some embodiments, the antibody comprises:

(a) a VH having at least 85% identity to a sequence selected from thegroup consisting of SEQ ID No.: 7 and SEQ ID No.: 146; and/or

(b) a VL having at least 85% identity to a sequence selected from thegroup consisting of SEQ ID No.: 8, SEQ ID No.: 136 and SEQ ID No.: 145.

The antibody can comprise a linker sequence, being or derived from SEQID No.: 9. In some embodiments, such antibody is an antibody fragmenthaving at least 85% sequence identity to a sequence selected from thegroup consisting of SEQ ID No.: 10, SEQ ID No.: 73 and SEQ ID No.: 82.

In one aspect the invention provides binding members that bind to IL-1beta and compete for binding with the antibodies described herein. Saidbinding member can be monovalent or multivalent. A preferred multivalentbinding member is bivalent. A multivalent binding member can bebispecific.

In one aspect, the invention provides an isolated nucleic acid sequenceencoding the antibody or the binding member disclosed herein.

In one aspect, a vector comprising said nucleic acid sequence isprovided.

In one aspect, the invention provides a host cell comprising the nucleicacid sequence above or the vector above.

In one aspect, a composition comprising the antibody above, the bindingmember above, the nucleic acid sequence above, the vector above or thehost cell above; and further a suitable carrier, diluent or excipient.The composition is preferably a pharmaceutical composition, comprising apharmaceutically acceptable carrier, diluent or excipient. Suchpharmaceutical composition is preferably in a form suitable for topical,intradermal, transdermal, intravenous, subcutaneous, intramuscular,parenteral, sublingual, buccal, oral, nasal, intranasal, rectal, localor ocular administration.

Further provided is a method of treating an IL-1 beta-mediated diseasecomprising administering to a subject in need thereof the pharmaceuticalcomposition above.

Also provided is the antibody above, the binding member above, thenucleic acid sequence above, the vector above or the host cell disclosedherein:

(i) for use in the treatment of an IL-1 beta-mediated disease;

(ii) for use in diagnostics;

(iii) for use in cosmetics; and/or

(iv) for detection purposes.

In still another aspect, the invention provides a method of producingthe antibody or the binding member described herein, either comprising:(i) the steps of cultivating the host cell above and recovering andpurifying the antibody fragment or the binding member, respectively; or(ii) the use of a cell-free system. Additionally or alternatively, themethod can comprise at least one step of chemical protein synthesis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of an ELISA to determine binding of DLX2323 torecombinant human (rh) IL-1 beta at various concentrations.

FIG. 2 is a graph depicting the results of DLX2323 binding to naturalhuman IL-1 beta in comparison to binding to rhIL-1 beta. Natural humanIL-1 beta was derived from supernatant of activated THP-1 cells.

FIGS. 3A and 3B show comparisons of DLX2323 with several commerciallyavailable IL-1 beta inhibitors for neutralization of rhIL-1 beta in ahuman fibroblast assay from two independent experiments. FIG. 3A showsthe results for DLX2323 and MAB201, and FIG. 3B summarizes data forDLX2323, rhIL-1 receptor antagonist (ra) and canakinumab (ILARIS®).

FIG. 4 shows the in vivo efficacy of DLX2323 in a human IL-1beta-induced mouse inflammation model.

FIG. 5 illustrates the definition of CDR-H1 as used herein.

FIG. 6 illustrates the results of an ELISA assay wherein cleared celllysates of DLX2323 variants expressed in E. coli cells bind bound tocoated rhIL-1 beta.

DETAILED DESCRIPTION

So that the invention may be more readily understood, certain terms arefirst defined. Unless otherwise defined within the specification, alltechnical and scientific terms used herein have their art-recognizedmeaning. Although similar or equivalent methods and materials to thosedescribed herein can be used in the practice or testing of theinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. The materials, methods, and examples are illustrative only andnot intended to be limiting.

Within the scope of the present invention, the term “antibody” refers tofull-length immunoglobulins as well as to fragments thereof. Suchfull-length immunoglobulins may be monoclonal, polyclonal, chimeric,humanized, veneered or human antibodies.

“Antibody fragments” comprise portions of a full-length immunoglobulinretaining the targeting specificity of said immunoglobulin. Many but notall antibody fragments lack at least partially the constant region (Fcregion) of the full-length immunoglobulin. In some embodiments, antibodyfragments are produced by digestion of the full-length immunoglobulin.An antibody fragment may also be a synthetic or recombinant constructcomprising parts of the immunoglobulin or immunoglobulin chains (seee.g. HOLLIGER, P. and Hudson, J. Engineered antibody fragments and therise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p.1126-1136). Examples of antibody fragments, without being limited to,include scFv, Fab, Fv, Fab′, F(ab′)₂ fragments, dAb, VHH, nanobodies,V(NAR) or minimal recognition units.

“Single chain variable fragments” or “single chain antibodies” or “scFv”are one type of antibody fragments. scFv are fusion proteins comprisingthe VH and VL of immunoglobulins connected by a linker. They thus lackthe constant Fc region present in full-length immunoglobulins, butretain the specificity of the original immunoglobulin.

A “binding member” as used herein refers to full-length immunoglobulins,antibody fragments, non-antibody scaffolds, and/or other bindingcompounds. Such binding member can be monovalent or multivalent, i.e.having one or more antigen binding sites. Non-limiting examples ofmonovalent binding members include scFv, Fab fragments, dAb, VHH,DARPins, affilins and nanobodies. A multivalent binding member can havetwo, three, four or more antigen binding sites whereby one or moredifferent antigens can be recognized. Full-length immunoglobulins,F(ab′)₂ fragments, bis-scFv and diabodies are non-limiting examples ofmultivalent binding members; in said exemplary multivalent bindingmembers, two binding sites are present, i.e. the binding member isbivalent.

In one embodiment, the multivalent binding member is bispecific, i.e.the binding member is directed against two different targets or twodifferent target sites on one target molecule. Bispecific antibodiesare, e.g., reviewed in MÜLLER, D. and Kontermann, R. E. Bispecificantibodies. Edited by DÜBEL, S. Weinheim: Wiley-VCH, 2007. ISBN3527314539. p. 345-378. In another embodiment, the multivalent bindingmember comprises more than two, e.g., three or four different bindingsites for three or four, respectively, different antigens. Such bindingmember is multivalent and multispecific, in particular tri- ortetra-specific, respectively.

“Non-antibody scaffolds” are antigen-binding polypeptides which are e.g.described in FIELDER, M. and Skerra, A. Non-antibody scaffolds. Editedby DÜBEL, S. Weinheim: Wiley-VCH, 2007. ISBN 3527314539. p. 467-500; orGILBRETH, R. N. and Koide, S. Structural insights for engineeringbinding proteins based on nonantibody scaffolds. Current Opinion inStructural Biology 2012, vol. 22, p. 413-420. Non-limiting examplesinclude affibodies, affilin molecules, AdNectin, Anticalin, DARPins,Knottin, Kunitz-type domain, Avimer, Tetranectin and trans-body.

“Binding compounds” are chemical or biological molecules that bind to atarget and that are not belonging to the class of full-lengthimmunoglobulins, antibody fragments and non-antibody scaffolds asdefined above. Examples of binding compounds, without being limited to,include macrolides (GUNDLURU, M. K. et al. Design, synthesis and initialbiological evaluation of a novel pladienolide analog scaffold.Medchemcomm. 2011, vol. 2, p. 904-908; PATERSON, I. et al. Totalsynthesis and biological evaluation of a series of macrocyclic hybridsand analogies of the antimitotic natural products dictyostatin,discodermolide and taxol. Chem Asian J. 2011, vol. 6, p. 459-473;MORITA, H. et al. Synthesis of unnatural alkaloid scaffolds byexploiting plant polyketide synthase. PNAS 2011, vol. 108, p.13504-13509), molecular imprinted polymers (HOSHINO, Y. et al.Recognition, neutralization and clearance of target peptides in theblood stream of living mice by molecular imprinted polymernanoparticles: a plastic antibody. Journal of the American ChemicalSociety, 2010, vol. 19, p. 664-6645), aptamers (STREHLITZ, B., et al.Aptamers for pharmaceuticals and their application in environmentalanalytics. Bioanalytical reviews 2012, vol. 4, p. 1-30; YE, M. et al.Generating Aptamers by Cell-SELEX for Applications in MolecularMedicine. International Journal of Molecular Sciences 2012, vol. 13, p.3341-3353), Spiegelmers (see e.g., MAASCH, C. et al.Polyethylenimine-Polyplexes of Spiegelmer NOX-A50 directed againstintracellular high mobility group protein A1 (HMGA1) reduce tumor growthin vivo. JBC 2010, vol. 285, p. 40012-40018), or peptides (cyclic orlinear; see, e.g., GOULD, A. et al. Cyclotides, a novel ultrastablepolypeptide scaffold for drug discovery. Curr Pharm Des. 2011, vol. 17,p. 4294-4307).

The “IC₅₀” or “half-maximum inhibitory concentration” is a measure ofantagonist drug potency and describes quantitatively the effectivenessof a compound to inhibit a biological or biochemical function. Thismeasure indicates how much of the compound is needed to inhibit by 50% acertain biological or biochemical process. Although no direct indicatorof affinity, both values are correlated and can be determined via theCheng-Prusoff equation (CHENG Y. and Prusoff W. H. Relationship betweenthe inhibition constant (Ki) and the concentration of inhibitor whichcauses 50 percent inhibition (ISO) of an enzymatic reaction. BiochemicalPharmacology 1973, vol. 22, p. 3099-3108; RAMMES, G., et al.Identification of a domain which affects kinetics and antagonisticpotency of clozapine at 5-HT3 receptors. PLOS one 2009, vol. 4, p. 1-14;ZHEN, J., et al. Concentration of receptor and ligand revisited in amodified receptor binding protocol for high-affinity radioligands: [³H]spiperone binding to D₂ and D₃ dopamine receptors. Journal ofNeuroscience Methods 2010, vol. 188, p. 32-38).

The term “IL-1 beta specific binding” as used herein describes that abinding member binds to IL-1 beta with higher affinity than to astructurally different antigen which does not comprise the IL-1 betaepitope to which the anti-IL-1 beta binding member binds. Specificbinding is reflected by a dissociation equilibrium constant (K_(D)) oflower than 1 micromolar. This constant can be determined, e.g. usingQuartz Crystal Microbalance (QCM) in an Attana instrument, or SurfacePlasmon Resonance (SPR) technology in a BIACORE instrument.

As used herein, “IL-1 beta” refers to the molecule as described in,e.g., Dinarello C. A., Treating inflammation by blocking interleukin-1in a broad spectrum of diseases. Nature reviews 2012, vol. 11, p.633-652. “hIL-1 beta” as used herein refers to human IL-1 beta. “rIL-1beta” refers to recombinant IL-1 beta. Recombinant IL-1 beta may or maynot have an amino terminal methionine residue, depending upon the methodby which it is prepared. “rhIL-1” beta refers to recombinant human IL-1beta. rhIL-1 beta may, e.g., be obtained from Peprotech, USA, cat. no.200-01B. IL-1 beta may also be obtained by isolation from biologicalsamples of human or non-human origin.

“Humanized” antibodies refer to antibodies comprising one or more,typically all six CDR regions of a non-human parent antibody or variantsthereof, and of which the framework is, e.g., (i) a human framework,potentially comprising one or more framework residues of the non-humanparent antibody, or (ii) a framework from a non-human antibody modifiedto increase similarity to naturally produced human frameworks. Methodsof humanizing antibodies are known in the art, see e.g. LEGER, O. andSaldanha, J. Antibody Drug Discovery. Edited by WOOD, C. London:Imperial College Press, 2011. ISBN 1848166281. p. 1-23.

“Framework” (FR) refers to the scaffold of the variable immunoglobulindomain, either the variable light chain (VL) or variable heavy chain(VH), embedding the respective CDRs. A VL and/or VH framework typicallycomprises four framework sections, FR1, FR2, FR3 and FR4, flanking theCDR regions. Thus, as known in the art, a VL has the general structure:(FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), whereas a VHhas the general structure:(FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H3)-(FR-H4).

“CDR” refers to the hypervariable regions of the antibody which mainlycontribute to antigen binding. Typically, an antigen binding sitecomprises six CDRs, embedded into a framework scaffold. Herein, the CDRsof the VL are referred to as CDR-L1, CDR-L2 and CDR-L3 whereas the CDRsof the VH are referred to as CDR-H1, CDR-H2 and CDR-H3. These can beidentified as described in KABAT, E. A., et al. Sequences of Proteins ofImmunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTHAND HUMAN SERVICES. NIH Publications, 1991. p. 91-3242. CDR-H1 as usedherein, however, differs from the Kabat definition in that it startswith position 27 and ends prior to position 36 (see FIG. 5 forillustration).

As used herein, the numbering system to identify amino acid residuepositions in the VH and VL of the antibody corresponds to the“AHo”-system described by HONEGGER, A. and Plückthun, A. Yet anothernumbering scheme for immunoglobulin variable domains: An automaticmodelling and analysis tool. Journal of Molecular Biology 2001, vol.309, p. 657-670. The publication further provides conversion tablesbetween the AHo and the Kabat system (KABAT, E. A., et al. Sequences ofProteins of Immunological Interest. 5th edition. Edited by U.S.DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p.91-3242).

An “isolated” antibody or nucleic acid is one being identified andseparated and/or recovered from at least one component of its naturalenvironment.

The term “identity” as used herein refers to the sequence match betweentwo proteins or nucleic acids. The protein or nucleic acid sequences tobe compared are aligned to give maximum identity, for example usingbioinformatics tools such as EMBOSS Needle (pair wise alignment). Whenthe same position in the sequences to be compared is occupied by thesame nucleobase or amino acid residue, then the respective molecules areidentical at that very position. Accordingly, the “percent identity” isa function of the number of matching positions divided by the number ofpositions compared and multiplied by 100%. For instance, if 6 out of 10sequence positions are identical, then the identity is 60%. The percentidentity between two protein sequences can, e.g., be determined usingthe Needleman and Wunsch algorithm (NEEDLEMAN, S. B. and Wunsch, C. D. Ageneral method applicable to the search for similarities in the aminoacid sequence of two proteins. Journal of Molecular Biology 1970, vol.48, p. 443-453) which has been incorporated into EMBOSS Needle, using aBLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an“end gap extend penalty” of 0.5. Two molecules having the same primaryamino acid or nucleic acid sequence are identical irrespective of anychemical and/or biological modification. For example, two antibodieshaving the same primary amino acid sequence but different glycosylationpatterns are identical by this definition. In case of nucleic acids, forexample, two molecules having the same sequence but different linkagecomponents such as thiophosphate instead of phosphate are identical bythis definition.

“Similar” protein sequences are those which, when aligned, share similaramino acid residues and most often, but not mandatorily, identical aminoacid residues at the same positions of the sequences to be compared.Similar amino acid residues are grouped by chemical characteristics ofthe side chains into families. Said families are described below for“conservative amino acid substitutions”. The “percent similarity”between sequences is the number of positions that contain identical orsimilar residues at the same sequence positions of the sequences to becompared divided by the total number of positions compared andmultiplied by 100%. For instance, if 6 out of 10 sequence positions haveidentical amino acid residues and 2 out of 10 positions contain similarresidues, then the sequences have 80% similarity. The similarity betweentwo sequences can e.g. be determined using EMBOSS Needle.

A “variant” refers to an amino acid or nucleic acid sequence whichdiffers from the parental sequence by virtue of addition (includinginsertions), deletion and/or substitution of one or more amino acidresidues or nucleobases while retaining at least one desired activity ofthe parent sequence disclosed herein. In the case of antibodies suchdesired activity may include specific antigen binding. Similarly, avariant nucleic acid sequence may be modified when compared to theparent sequence by virtue of addition, deletion and/or substitution ofone or more nucleobases, but the encoded antibody retains the desiredactivity as described above. Variants may be naturally occurring, suchas allelic or splice variants, or may be artificially constructed.

As used herein, the term “conservative modifications” refers tomodifications that are physically, biologically, chemically orfunctionally similar to the corresponding reference, e.g., has a similarsize, shape, electric charge, chemical properties, including the abilityto form covalent or hydrogen bonds, or the like. Such conservativemodifications include, but are not limited to, one or more nucleobasesand amino acid substitutions, additions and deletions.

For example, conservative amino acid substitutions include those inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. For example, amino acid residues beingnon-essential with regard to binding to an antigen can be replaced withanother amino acid residue from the same side chain family, e.g. serinemay be substituted for threonine. Amino acid residues are usuallydivided into families based on common, similar side-chain properties,such as:

-   -   1. nonpolar side chains (e.g., glycine, alanine, valine,        leucine, isoleucine, methionine),    -   2. uncharged polar side chains (e.g., asparagine, glutamine,        serine, threonine, tyrosine, proline, cysteine, tryptophan),    -   3. basic side chains (e.g., lysine, arginine, histidine,        proline),    -   4. acidic side chains (e.g., aspartic acid, glutamic acid),    -   5. beta-branched side chains (e.g., threonine, valine,        isoleucine) and    -   6. aromatic side chains (e.g., tyrosine, phenylalanine,        tryptophan, histidine).        A conservative substitution may also involve the use of a        non-natural amino acid.

Non-conservative substitutions, i.e. exchanging members of one familyagainst members of another family, may lead to substantial changes,e.g., with respect to the charge, dipole moment, size, hydrophilicity,hydrophobicity or conformation of the binding member, which may lead toa significant drop in the binding activity, in particular if amino acidsare affected that are essential for binding to the target molecule. Anon-conservative substitution may also involve the use of a non-naturalamino acid.

Conservative and non-conservative modifications can be introduced intoparental binding members by a variety of standard techniques known inthe art, such as combinatorial chemistry, site-directed DNA mutagenesis,PCR-mediated and/or cassette mutagenesis, peptide/protein chemicalsynthesis, chemical reaction specifically modifying reactive groups inthe parental binding member. The variants can be tested by routinemethods for their chemical, biological, biophysical and/or biochemicalproperties.

Nucleic acid hybridization reactions can be performed under conditionsof different stringency. “Stringent conditions” are widely known andpublished in the art. Typically, during the hybridization reaction aSSC-based buffer can be used in which SSC is 0.15 M NaCl and 15 mMcitrate buffer having a pH of 7.0. Increasing buffer concentrations andthe presence of a denaturing agent increase the stringency of thehybridization step. For example, high stringency hybridizationconditions can involve the use of: (i) 50% (vol/vol) formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C. with washesat 42° C. in 0.2×SSC and 0.1% SDS; (ii) 50% (vol/vol) formamide with0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mMsodium citrate at 42° C.; or (iii) 10% dextran sulfate, 2×SSC, and 50%formamide at 55° C., followed by a high-stringency wash consisting of0.1×SSC containing EDTA at 55° C. Additionally or alternatively, one,two or more washing steps using wash solutions of low ionic strength andhigh temperature can be included in the hybridization protocol using,for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

In a first aspect, the invention provides a monovalent antibody fragmentbinding IL-1 beta which inhibits the biological effect of human IL-1beta with an IC₅₀ of lower than 50 pM. Said IC₅₀ is preferably lowerthan about 40 pM, more preferably lower than about 30, 20, 10, 5, 4, 3,2 or 1 pM.

Preferably, said monovalent antibody fragment has a molecular weight ofabout 50 kDa or lower, such as about 45 kDa, 40 kDa, 35 kDa or lower,preferably about 25 kDa, such as 23, 24, 25, 26, or 27 kDa.

In one aspect, the invention provides an antibody, comprising:

(a) at least one of the VH CDR sequences CDR-H1, CDR-H2 or CDR-H3 as setforth in SEQ ID Nos.: 1, 2 and 3, respectively, or variants thereofand/or

(b) at least one of the VL CDR sequences CDR-L1, CDR-L2 or CDR-L3 as setforth in SEQ ID Nos.: 4, 5, and 6, respectively, or variants thereof.

Such antibody may comprise:

(a) at least one of the VH CDR sequences CDR-H1, CDR-H2 or CDR-H3 as setforth in SEQ ID Nos.: 155, 156 and 157, respectively, or variantsthereof and/or

(b) at least one of the VL CDR sequences CDR-L1, CDR-L2 or CDR-L3,

(i) as set forth in SEQ ID Nos.: 158, 159 and 160, respectively, orvariants thereof, or

(ii) as set forth in SEQ ID Nos.: 161, 162 and 163, respectively, orvariants thereof.

Preferably, the antibody comprises at least the CDR-H3 of SEQ ID No.: 3and the CDR-L3 of SEQ ID No.: 6 or SEQ ID No.: 157, or a variantthereof, respectively. Even more preferably, said antibody comprises allsix CDRs of:

(i) SEQ ID Nos.: 1 to 6 or variants thereof;

(ii) SEQ ID Nos.: 155 to 160 or variants thereof or

(iii) SEQ ID Nos.: 155 to 157 and SEQ ID Nos.: 161 to 163 or variantsthereof.

Such antibody has a very high inhibitory potency against human IL-1 betawith an IC₅₀ of lower than 50 pM, more preferably lower than about 40pM, 30, 20, 10, and even more preferably lower than 5 pM and mostpreferably about 1 pM and lower.

Preferably, the antibody has an inhibitory potency against human IL-1beta with an IC₅₀ of at least 2 pM, more preferably of at least 1 pM.

The IC₅₀ can, e.g., be determined using a cell based potency assay. Inone embodiment, the IC₅₀ value above is determined by inhibiting theIL-1 beta induced release of IL-6 from human fibroblasts. Such assay isbased on the observation that fibroblasts stimulated with IL-1 betarelease IL-6. In the presence of IL-1 beta inhibiting antibodies, theconcentration of released IL-6 is reduced. In a preferred embodiment,Normal Human Dermal Fibroblasts (NHDF-Neo, e.g., obtainable from LonzaWalkersville USA, cat. no. CC-2509) cells are used. Upon incubation witha mixture of hIL-1 beta and the antibody of interest, supernatants areharvested and examined by an IL-6 ELISA such as the R&D Systems HumanIL-6 DuoSet ELISA kit (R&D Systems, cat. no. DY206). In one embodiment,the assay is the IL-1 beta neutralization assay as described in example3. Preferably, the IC₅₀ value is the mean value obtained of at leastthree independent repetitions of such assay.

The antibody described herein may be a full-length immunoglobulin or anantibody fragment, such as a Fab, Fab′, F(ab′)₂, scFv, Fv fragment,nanobody, VHH or minimal recognition unit.

In a preferred embodiment the antibody and in particular the monovalentantibody fragment above is a scFv. The VH and VL domains can beconnected in either orientation, VL-linker-VH or VH-linker-VL, by aflexible linker. In a preferred embodiment, the orientation isVL-linker-VH, i.e. the light chain variable region being at theN-terminal end and the heavy chain variable region being at theC-terminal end of the polypeptide.

The antibody is preferably humanized. Such humanized antibody may, e.g.,comprise in the variable light chain the FR-L1 of SEQ ID No.: 18, theFR-L2 of SEQ ID No.: 19, the FR-L3 of SEQ ID No.: 20 and/or the FR-L4 ofSEQ ID No.: 21 or variants thereof. Additionally or alternatively, thehumanized antibody can comprise the heavy chain variable frameworkregion FR-H1 of SEQ ID No.: 22, 26 or 30; the heavy chain variableframework region FR-H2 of SEQ ID No.: 23, 27 or 31; the heavy chainvariable framework region FR-H3 of SEQ ID No.: 24, 28 or 32; and/or theheavy chain variable framework region FR-H4 of SEQ ID No.: 25, 29 or 33.

Thus, in a preferred embodiment, the antibody comprises the VH sequenceof SEQ ID No.: 7 or SEQ ID No.: 146, or a variant thereof, respectively.Such variant has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity toSEQ ID No.: 7 or SEQ ID No.: 146. Examples of such variant VH sequencesinclude, without being limited to, SEQ ID No.: 121, SEQ ID No.: 122, SEQID No.: 124, SEQ ID No.: 126, SEQ ID No.: 128, SEQ ID No.: 130, SEQ IDNo.: 132, SEQ ID No.: 134, SEQ ID No.: 142, SEQ ID No.: 144, SEQ ID No.:146, SEQ ID No.: 148, SEQ ID No.: 150 or SEQ ID No.: 152.

Additionally or alternatively, the antibody disclosed herein comprisesthe VL sequence selected from the group consisting of SEQ ID No.: 8, SEQID No.: 136 and SEQ ID No.: 145, or a variant thereof, respectively.Such variant has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity toSEQ ID No.: 8, SEQ ID No.: 136 or SEQ ID No.: 145. Examples of suchvariant VL sequences include, without being limited to, SEQ ID No.: 123,SEQ ID No.: 125, SEQ ID No.: 127, SEQ ID No.: 129, SEQ ID No.: 131, SEQID No.: 133, SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ IDNo.: 139 or SEQ ID No.: 153.

In one embodiment, the antibody comprises a VH sequence having at least85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%and most preferably 100% sequence similarity to SEQ ID No.: 7 or SEQ IDNo.: 146. Additionally or alternatively, the antibody comprises a VLsequence having at least 85%, more preferably 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and most preferably 100% sequence similarity toSEQ ID No.: 8, SEQ ID No.: 136 or SEQ ID No.: 145.

In a much preferred embodiment, the antibody comprises the VH as setforth in to SEQ ID No.: 7 and the VL as set forth in SEQ ID No.: 8. Theframework sequences of both SEQ ID No.: 7 and SEQ ID No.: 8 are derivedfrom a human immunoglobulin described in WO 03/097697 A (ESBATech AG).Its VH and VL framework sequences have been modified for humanizationand stabilization of rabbit antibodies, see, e.g., WO 2009/155726 A(ESBATech, AN ALCON BIOMEDICAL RESEARCH UNIT LLC); BORRAS, L., et al.Generic approach for the generation of stable humanized single-chain Fvfragments from rabbit monoclonal antibodies. Journal of BiologicalChemistry 2010, vol. 285, no. 12, p. 9054-9066. In one embodiment, theVL framework of the antibody disclosed herein comprises SEQ ID Nos.:18-21 or variants thereof. Additionally or alternatively, the VHframework of the antibody comprises SEQ ID Nos.: 22-25, SEQ ID Nos.:26-29 or SEQ ID Nos.: 30-33 or variants thereof, respectively.

In another preferred embodiment, the antibody comprises the VH as setforth in to SEQ ID No.: 146 and the VL as set forth in SEQ ID No.: 8 orin SEQ ID No.: 145.

In another preferred embodiment, the antibody comprises the VH as setforth in to SEQ ID No.: 146 and the VL as set forth in SEQ ID No.: 136.

The antibody, in particular in case of a scFv, may comprise a linkersequence. Such linker sequence has typically ten to about 25 aminoacids. Usually, such linker peptide is rich in glycines, which conferflexibility, as well as serines and/or threonines for improvedsolubility. In a preferred embodiment, a (GGGGS)₄ linker (SEQ ID No.: 9)or a variant thereof is used. Variations of said motif having three tofive repeats may also be used. Further suitable linkers are described,e.g., in ALFTHAN, K. Properties of a single-chain antibody containingdifferent linker peptides. Protein Engineering 1995, vol. 8, no. 7, p.725-731.

In certain embodiments variants of the antibodies provided herein arecontemplated. For example, it may be desirable to improve antigenbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC), to increase stability orsolubility, to decrease immunogenicity and/or to alter other biological,biochemical or biophysical properties of the antibody. In someembodiments the variant does not show any improvement over the parentantibody.

Variants of the antibodies provided herein may be prepared by proteinand/or chemical engineering, introducing appropriate modifications intothe nucleic acid sequence encoding the antibody, or by protein/peptidesynthesis. Any combination(s) of deletions, substitutions, additions andinsertions can be made to the framework or to the CDRs, provided thatthe generated antibody possesses the desired characteristics for whichit can be screened using appropriate methods. Of particular interest aresubstitutions, preferably conservative substitutions as described above.Preferred conservative substitutions include:

-   -   1. Substituting alanine (A) by valine (V);    -   2. Substituting arginine (R) by lysine (K);    -   3. Substituting asparagine (N) by glutamine (Q);    -   4. Substituting aspartic acid (D) by glutamic acid (E);    -   5. Substituting cysteine (C) by serine (S);    -   6. Substituting glutamic acid (E) by aspartic acid (D);    -   7. Substituting glycine (G) by alanine (A);    -   8. Substituting histidine (H) by arginine (R) or lysine (K);    -   9. Substituting isoleucine (I) by leucine (L);    -   10. Substituting methionine (M) by leucine (L);    -   11. Substituting phenylalanine (F) by tyrosine (Y);    -   12. Substituting proline (P) by alanine (A);    -   13. Substituting serine (S) by threonine (T);    -   14. Substituting tryptophan (W) by tyrosine (Y);    -   15. Substituting phenylalanine (F) by tryptophan (W); and/or    -   16. Substituting valine (V) by leucine (L) and vice versa.

The antibody described herein may comprise one or more, such as two,three, four, five, six, seven, eight, nine, ten, eleven, twelve or moreof such conservative substitutions.

Non-conservative substitutions may lead to more substantial changes,e.g., with respect to the charge, dipole moment, size, hydrophilicity,hydrophobicity or conformation of the polypeptide. In one embodiment theantibody comprises one or more, such as two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or more of such non-conservativesubstitutions.

Modifications may be present in the CDRs or in the framework sequences.For example, the CDRs provided herein may comprise one, two, three,four, five or even more modifications. For example, the CDR-L1, CDR-L2and CDR-L3 sequences taken as a whole are at least 75%, preferably atleast 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or more preferably 99% identical to the CDRs provided herein,in particular to (i) SEQ ID Nos.: 4, 5 and 6, or to (ii) SEQ ID Nos.:161, 162 and 163. Additionally or alternatively, the CDR-H1, CDR-H2 andCDR-H3 sequences taken as a whole are at least 80%, preferably at least81%, 82%, 83%, 84%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% ormore preferably 99% identical to the CDRs provided herein, in particularto (i) SEQ ID Nos.: 1, 2 and 3, or to (ii) SEQ ID Nos.: 155, 156 and157.

In one embodiment the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3taken as a whole are at least 85%, preferably 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or more preferably 99% similar to the CDRs providedherein. Additionally or alternatively, the CDR-L1, CDR-L2, CDR-L3,CDR-H1, CDR-H2 and CDR-H3 taken as a whole are at least 85%, preferably90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99%similar to the CDRs provided herein.

Therefore, a variant may, e.g., comprise one, two, three, four or fivesubstitutions in SEQ ID No.: 4. Much preferred are substitutions atpositions marked with X in SEQ ID No.: 14. The variant may, e.g.,comprise:

-   -   (i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 32        of the variable light chain;    -   (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), serine (S), threonine (T), valine        (V), tryptophan (W), tyrosine (Y) at AHo position 33 of the        variable light chain; and/or    -   (iii) glutamic acid (E), phenylalanine (F), glycine (G),        methionine (M), asparagine (N), glutamine (Q), serine (S),        tryptophan (W), tyrosine (Y) at AHo position 40 of the variable        light chain.

Additionally or alternatively, a variant comprises one, two, three, orfour substitutions in SEQ ID No.: 5. Much preferred are substitutions atpositions marked with X in SEQ ID No.: 15. Such variant may, e.g.,comprise:

-   -   (i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), tryptophan (W), tyrosine (Y) at AHo position 58 of the        variable light chain; and/or    -   (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 69        of the variable light chain.

Additionally or alternatively, a variant comprises one, two, three,four, five or six substitutions in SEQ ID No.: 6. Much preferred aresubstitutions at positions marked with X in SEQ ID No.: 16. For example,such variant may comprise:

-   -   (i) alanine (A), cysteine (C), isoleucine (I), asparagine (N),        serine (S), threonine (T), valine (V) at AHo position 109 of the        variable light chain;    -   (ii) alanine (A), glycine (G), proline (P), serine (S) at AHo        position 111 of the variable light chain;    -   (iii) alanine (A), cysteine (C), aspartic acid (D), glutamic        acid (E), phenylalanine (F), glycine (G), histidine (H),        isoleucine (I), lysine (K), leucine (L), methionine (M),        asparagine (N), proline (P), glutamine (Q), arginine (R), serine        (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y) at        AHo position 112 of the variable light chain;    -   (iv) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position        135 of the variable light chain; and/or    -   (v) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), leucine (L), methionine (M), asparagine (N), proline (P),        glutamine (Q), arginine (R), serine (S), threonine (T), valine        (V), tryptophan (W), tyrosine (Y) at AHo position 136 of the        variable light chain.

Additionally or alternatively, a variant comprises one, two, three, orfour substitutions in SEQ ID No.: 1 or in SEQ ID No.: 155. Muchpreferred are substitutions at positions marked with X in SEQ ID No.:11. Such variant may, e.g., comprise:

-   -   (i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position 33        of the variable heavy chain; and/or    -   (ii) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        glutamine (Q), arginine (R), serine (S), threonine (T), valine        (V), tryptophan (W), tyrosine (Y) at AHo position 39 of the        variable heavy chain.

Additionally or alternatively, a variant comprises one, two, three,four, five or six substitutions in SEQ ID No.: 2 or in SEQ ID No.: 156.Much preferred are substitutions at positions marked with X in SEQ IDNo.: 12. For example, the variant may comprise:

-   -   (i) alanine (A), cysteine (C), glycine (G), methionine (M) or        tyrosine (Y) at AHo position 59 of the variable heavy chain;    -   (ii) aspartic acid (D), asparagine (N) or proline (P) at AHo        position 60 of the variable heavy chain; and/or    -   (iii) alanine (A), aspartic acid (D), glutamic acid (E), glycine        (G), phenylalanine (F), histidine (H), isoleucine (I), lysine        (K), leucine (L), methionine (M), asparagine (N), proline (P),        serine (S), threonine (T), tryptophan (W) or tyrosine (Y) at AHo        position 69 of the variable heavy chain.

Additionally or alternatively, a variant comprises one, two, three,four, five, six, seven, eight, nine, ten or eleven substitutions in SEQID No.: 3 or in SEQ ID No.: 157. Much preferred are substitutions atpositions marked with X in SEQ ID No.: 13. Such variant may, e.g.,comprise:

-   -   (i) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        glutamine (Q), arginine (R), serine (S), threonine (T), valine        (V), tryptophan (W), tyrosine (Y) at AHo position 110 of the        variable heavy chain;    -   (ii) alanine (A), cysteine (C), aspartic acid (D), phenylalanine        (F), glycine (G), histidine (H), isoleucine (I), lysine (K),        methionine (M), asparagine (N), proline (P), glutamine (Q),        arginine (R), serine (S), threonine (T), valine (V), tryptophan        (W), tyrosine (Y) at AHo position 111 of the variable heavy        chain;    -   (iii) alanine (A), cysteine (C), phenylalanine (F), histidine        (H), isoleucine (I), leucine (L), methionine (M), asparagine        (N), glutamine (Q), serine (S), threonine (T), valine (V),        tyrosine (Y) at AHo position 112 of the variable heavy chain;    -   (iv) phenylalanine (F) or isoleucine (I) at AHo position 113 of        the variable heavy chain;    -   (v) alanine (A), cysteine (C), glutamic acid (E), glycine (G),        serine (S), threonine (T), valine (V) at AHo position 114 of the        variable heavy chain;    -   (vi) alanine (A), glycine (G), methionine (M) or asparagine (N)        at AHo position 115 of the variable heavy chain;    -   (vii) alanine (A), aspartic acid (D), glutamic acid (E),        histidine (H), asparagine (N), serine (S), threonine (T) at AHo        position 135 of the variable heavy chain;    -   (viii) alanine (A), cysteine (C), phenylalanine (F), glycine        (G), histidine (H), isoleucine (I), leucine (L), methionine (M),        asparagine (N), glutamine (Q), serine (S), threonine (T), valine        (V), tryptophan (W), tyrosine (Y) at AHo position 136 of the        variable heavy chain;    -   (ix) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position        137 of the variable heavy chain; and/or    -   (x) alanine (A), cysteine (C), aspartic acid (D), glutamic acid        (E), phenylalanine (F), glycine (G), histidine (H), isoleucine        (I), lysine (K), leucine (L), methionine (M), asparagine (N),        proline (P), glutamine (Q), arginine (R), serine (S), threonine        (T), valine (V), tryptophan (W), tyrosine (Y) at AHo position        138 of the variable heavy chain.

A particularly preferred type of variant is one where one or more entireCDRs are replaced. Typically, the CDR-H3 and CDR-L3 contribute mostsignificantly to antigen binding. For example, the entire CDR-L1,CDR-L2, CDR-H1 and/or CDR-H2 may be replaced by a different CDR ofnatural or artificial origin. In some embodiments, one or more CDRs arereplaced by an alanine-cassette.

In one embodiment, the variant described herein has at least 85%, morepreferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and mostpreferably 100% sequence identity to a sequence selected from the groupconsisting of SEQ ID No.: 10, SEQ ID No.: 73 and SEQ ID No.: 82.

In one embodiment, the variant described herein has at least 85%, morepreferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and mostpreferably 100% sequence similarity to SEQ ID No.: 10, SEQ ID No.: 73and SEQ ID No.: 82.

Additionally or alternatively, the VH of the antibody comprisessolubility enhancing point mutations. WO2009/155725 (ESBATech, aNovartis company) describes a motif, which has proven to increase theoverall solubility of the antibody. The residues are placed at positionslocated in the interface of the variable domain and the constant domainof an antibody and stabilize antibody fragments, in particular scFv,lacking the constant domain. In particular, one, preferably all three ofthe following residues are present:

(i) serine (S) at heavy chain amino acid position 12 (according to AHonumbering);

(ii) serine (S) or threonine (T) at heavy chain amino acid position 103(according to AHo numbering); and/or

(iii) serine (S) or threonine (T) at heavy chain amino acid position 144(according to AHo numbering).

In a preferred embodiment the antibody has a serine at VH position 12; aserine at VH position 103; and a threonine at VH position 144 (all AHonumbering).

Thus, in one embodiment the antibody disclosed herein comprises the VHframework sequences of SEQ ID Nos.: 30-33 or variants thereof.

Preferably, a variant antibody as used herein:

(i) retains specific binding to IL-1 beta, in particular to hIL-1 beta;

(ii) has a potency (IC₅₀) with regard to inhibiting the biologicaleffect of human IL-1 beta of lower than 500 pM, preferably lower than400 pM, 300 pM, 200 pM, 100 pM, 50 pM, more preferably of lower than 25pM;

(iii) is cross-reactive with cynomolgus IL-1 beta, rhesus monkey IL-1beta and/or rat IL-1 beta; and/or

(iv) competes with the antibody disclosed herein for binding to IL-1beta, preferably human IL-1 beta, cynomolgus IL-1 beta, rhesus monkeyIL-1 beta and/or rat IL-1 beta, most preferably hIL-1 beta.

In one embodiment, the variant comprises a VL sequence selected from thegroup consisting of SEQ ID No.: 96, SEQ ID No.: 97, SEQ ID No.: 98, SEQID No.: 99, SEQ ID No.: 100, SEQ ID No.: 101, SEQ ID No.: 102, SEQ IDNo.: 103, SEQ ID No.: 104, SEQ ID No.: 105, SEQ ID No.: 123, SEQ ID No.:125, SEQ ID No.: 127, SEQ ID No.: 129, SEQ ID No.: 131, SEQ ID No.: 133,SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ ID No.: 139, SEQID No.: 141, SEQ ID No.: 143, SEQ ID No.: 145, SEQ ID No.: 147, SEQ IDNo.: 149, SEQ ID No.: 151 and SEQ ID No.: 153.

Additionally or alternatively, the variant comprises a VH sequenceselected from the group consisting of SEQ ID No.: 106, SEQ ID No.: 107,SEQ ID No.: 108, SEQ ID No.: 109, SEQ ID No.: 110, SEQ ID No.: 111, SEQID No.: 112, SEQ ID No.: 113, SEQ ID No.: 114, SEQ ID No.: 115, SEQ IDNo.: 116, SEQ ID No.: 117, SEQ ID No.: 118, SEQ ID No.: 119, SEQ ID No.:120, SEQ ID No.: 121, SEQ ID No.: 122, SEQ ID No.: 124, SEQ ID No.: 126,SEQ ID No.: 128, SEQ ID No.: 130, SEQ ID No.: 132, SEQ ID No.: 134, SEQID No.: 138, SEQ ID No.: 140, SEQ ID No.: 142, SEQ ID No.: 144, SEQ IDNo.: 146, SEQ ID No.: 148, SEQ ID No.: 150, SEQ ID No.: 152.

Variants may also be prepared by chain shuffling of light and heavychains. A single light chain can be combined with a library of heavychains to yield a library of variants. In one embodiment, said singlelight chain is selected from the group of VL sequences recited aboveand/or said library of heavy chains comprises one or more of the VHsequences recited above. Likewise, a single heavy chain can be combinedwith a library of light chains. Preferably, said single heavy chain isselected from the group of VH sequences recited above and/or saidlibrary of light chains comprises one or more of the VL sequencesrecited above.

In one embodiment, the variant comprises the VL of SEQ ID No.: 135and/or the VH of SEQ ID No.: 7, SEQ ID No.: 142, SEQ ID No.: 146, SEQ IDNo.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ IDNo.: 67, SEQ ID No.: 85, SEQ ID No.: 86, SEQ ID No.: 87 or SEQ ID No.:88.

In one embodiment, the variant comprises the VL of SEQ ID No.: 136and/or the VH of SEQ ID No.: 7, SEQ ID No.: 142, SEQ ID No.: 146, SEQ IDNo.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ IDNo.: 68, SEQ ID No.: 81, SEQ ID No.: 82, SEQ ID No.: 83 or SEQ ID No.:84.

In one embodiment, the variant comprises the VL of SEQ ID No.: 137and/or the VH of SEQ ID No.: 7, SEQ ID No.: 138, SEQ ID No.: 142, SEQ IDNo.: 146, SEQ ID No.: 150 or SEQ ID No.: 152. Preferably, the variantcomprises SEQ ID No.: 69, SEQ ID No.: 92, SEQ ID No.: 93, SEQ ID No.: 94or SEQ ID No.: 95.

In one embodiment, the variant comprises the VL of SEQ ID No.: 139and/or the VH of SEQ ID No.: 140, SEQ ID No.: 142, SEQ ID No.: 146, SEQID No.: 150 or SEQ ID No.: 152. Preferably, the variant comprises SEQ IDNo.: 70, SEQ ID No.: 77, SEQ ID No.: 78, SEQ ID No.: 79 or SEQ ID No.:80.

In one embodiment, the variant comprises the VL of SEQ ID No.: 141and/or the VH of SEQ ID No.: 142. Preferably, the variant comprises SEQID No.: 71.

In one embodiment, the variant comprises the VL of SEQ ID No.: 143and/or the VH of SEQ ID No.: 144. Preferably, the variant comprises SEQID No.: 72.

In one embodiment, the variant comprises the VL of SEQ ID No.: 145and/or the VH of SEQ ID No.: 146. Preferably, the variant comprises SEQID No.: 73.

In one embodiment, the variant comprises the VL of SEQ ID No.: 147and/or the VH of SEQ ID No.: 148. Preferably, the variant comprises SEQID No.: 74.

In one embodiment, the variant comprises the VL of SEQ ID No.: 149and/or the VH of SEQ ID No. 150. Preferably, the variant comprises SEQID No.: 75.

In one embodiment, the variant comprises the VL of SEQ ID No.: 151and/or the VH of SEQ ID No. 152. Preferably, the variant comprises SEQID No.: 76.

In one embodiment, the variant comprises the VL of SEQ ID No.: 8 and/orthe VH of SEQ ID No.: 121 or of SEQ ID No.: 122. Preferably, the variantcomprises SEQ ID No.: 59 or SEQ ID No.: 60.

In one embodiment, the variant comprises the VL of SEQ ID No.: 153and/or the VH of SEQ ID No.: 142, SEQ ID No.: 146 or SEQ ID No.: 152.Preferably, the variant comprises SEQ ID No. 89, SEQ ID No.: 90 or 91.

In one embodiment, the variant comprises the VL of SEQ ID No.: 8 and/orthe VH of SEQ ID No.: 121, SEQ ID No.: 122, SEQ ID No.: 142, SEQ ID No.:144, SEQ ID No.: 146, SEQ ID No.: 148, SEQ ID No.: 150 or SEQ ID No.:152.

In one embodiment, the variant comprises the VH of SEQ ID No.: 7 and/orthe VL of SEQ ID No.: 135, SEQ ID No.: 136, SEQ ID No.: 137, SEQ ID No.:139 or SEQ ID No.: 153.

In one embodiment, the variant comprises a sequence selected from thegroup consisting of SEQ ID No.: 34 to 95 and SEQ ID No.: 154.

A binding member can comprise any of the VL and/or the VH sequencesmentioned above. Binding members having a single domain format, such asa nanobody or a VHH, comprise only one of either the VL or VH sequencesmentioned above, preferably the VH sequence. Multivalent bindingmembers, in particular F(ab′)₂ fragments, bis-scFv or diabodies,preferably bispecific binding members, may comprise one or more of theVL sequences mentioned above and/or one or more of the VH sequencesmentioned above.

The antibodies of the instant invention are particularly stable. As usedherein the term “stability” refers to the biophysical property of theantibody to remain monomeric in solution after prolonged incubationand/or incubation at elevated temperature. Unstable antibodies tend todimerize or oligomerize and even precipitate, thereby decreasingshelf-life and becoming less suitable for pharmaceutical applications.

The antibodies provided herein and in particular the monovalent antibodyfragment above remain monomeric at least to 75%, preferably at least to80%, 85%, and most preferably to 93% after being incubated for 1 monthat 37° C. at a concentration of 1 mg/ml in PBS at pH 7.2. Additionallyor alternatively, the antibody remains monomeric at least to 90%,preferably at least to 92%, 94%, 96%, 98% more preferably to 100% after1 month at room temperature at a concentration of 1 mg/ml in PBS at pH7.2.

The degree of monomers can, e.g., be determined by SEC-HPLC (SizeExclusion Chromatography-High-Performance Liquid Chromatography). Asuitable mobile phase for such testing is, e.g., PBS at pH 7.2. Themonomer content can be quantified by peak integration of the UV280signal measured during the protein chromatography. A suitable system is,e.g., a Dionex Summit HPLC controlled by CHROMELEON® 6.5 software thatalso allows for subsequent chromatogram analysis and peakquantification.

The antibodies disclosed herein and in particular the monovalentantibody fragment above are also stable at higher concentrations, forexample, they remain monomeric at least to 50%, preferably at least to55%, 60%, 65%, 70% and most preferably to 75% after being incubated for2 weeks at room temperature and/or 4° C. at a concentration of about 50mg/ml in PBS at pH 7.2.

Moreover, the antibodies provided herein and in particular themonovalent antibody fragment above are particularly soluble and cantherefore be highly concentrated without precipitation due to aggregateformation. Preferably, the antibodies can be concentrated in PBS at pH7.2 to a concentration of more than 20 mg/ml without precipitation, morepreferably to a concentration of 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/mland most preferably to 70 mg/ml in PBS at pH 7.2.

In a much preferred embodiment, the antibody has a melting temperatureof about 60° C. as determined by differential scanning fluorimetry(DSF), preferably 65° C., 70° C., 71° C., 72° C., 73° C. and mostpreferably 74° C. This method is based on the properties of certain dyesbeing fluorescent only in a hydrophobic environment. For example,protein unfolding can be detected as an increase in fluorescence uponbinding of the dye SYPRO® Orange to a heat-denatured protein (NIESEN F.H. et al. The use of differential scanning fluorimetry to detect ligandinteractions that promote protein stability. Nature Protocols 2007, vol.2, p. 2212-2221). The stability of a protein can thus be analyzed bythermal denaturation.

The antibody has preferably a theoretical isoelectric point (pI) in therange of 5 to 10, preferably 7 to 9, most preferably about 8.3. Thetheoretical pI can, for example, be calculated by using the ProtParamtool on the ExPASy Server (see also GASTEIGER E. et al. ProteinIdentification and Analysis Tools on the ExPASy Server. (In) TheProteomics Protocols Handbook. Edited by WALKER J. M. Totowa: HumanaPress Inc., 2005. ISBN 9781588295934. p. 571-607).

The antibody can be cross-reactive with IL-1 beta from non-humanspecies, such as, without being limited to, cynomolgus IL-1 beta, rhesusmonkey IL-1 beta, rat IL-1 beta, murine IL-1 beta, canine IL-1 beta,feline IL-1 beta, marmoset IL-1 beta, swine IL-1 beta and/or guinea pigIL-1 beta. Preferably, the antibody is cross-reactive with cynomolgusIL-1 beta (e.g., recombinantly produced and available from SinoBiological Inc., cat. no. 90010-CNAE), rhesus monkey IL-1 beta (e.g.,recombinantly produced and available from R&D Systems, cat. no.1318-RL/CF) and/or rat IL-1 beta (e.g., recombinantly produced andavailable from Peprotech, cat. no. 400-01B).

Preferably, there is no residual activity of IL-1 beta when beingneutralized with the antibody disclosed herein in an in vivo and/or anin vitro setting, i.e. the antibody completely inhibits the action ofIL-1 beta. “No residual activity” as used herein refers to lower than 2%of the potency assay signal corresponding to the IL-6 release from humanfibroblasts induced by 10 pg/ml of IL-1 beta, preferably the assay asdescribed in example 3, in presence of 60 ng/ml of the antibodydescribed herein when compared to antibodies of non-relevant specificityor vehicle control at the same concentration.

The invention also provides a binding member competing with theantibodies disclosed herein for binding to human IL-1 beta.

As used herein, the term “competing” refers to the competition betweenbinding members for binding to the same target. Competition can bedetermined by competitive binding assays in which the binding member ofinterest prevents or inhibits or reduces specific binding of theantibodies disclosed herein to a common antigen (here, hIL-1 beta or afragment thereof). Such competitive binding assays are known in the artand include, without being limited to, solid phase direct or indirectradioimmunoassay (MA) and solid phase direct or indirect enzymeimmunoassay (EIA). Typically, such assay involves the use of purifiedantigen bound to a solid surface, a binding member to be tested and thereference antibody as described herein. Competitive inhibition ismeasured by determining the amount of either: (i) the reference antibodybound to the solid surface in the presence of the binding member to betested, or (ii) the binding member to be tested bound to the solidsurface in the presence of the reference antibody. A competing bindingmember may bind: (i) to the same epitope as the antibody, (ii) to anoverlapping epitope, or (iii) to a different epitope on the same targetmolecule but sterically hindering binding of the antibody to its target.

Usually, when a competing binding member is present in excess, it willreduce specific binding of the antibody as described herein to IL-1 betaby at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or75% or more. Preferably, binding of the antibody is reduced by at least80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

In one embodiment, the competing binding member binds to hIL-1 beta withan affinity K_(D) of at least about 1 pM, 10 pM, 100 pM, 500 pM, 1 nM,10 nM.

In one embodiment, the binding member is monovalent, such as a scFv or aFab fragment. In another embodiment, the binding member is multivalent.Such multivalent molecule can be bivalent (such as a full-lengthimmunoglobulin or a F(ab′)₂ fragment) or comprises at least three targetbinding sites.

The multivalent binding member can be a bispecific antibody such as adiabody, a single-chain diabody or a tandem scFv (see, e.g., KONTERMANN,R. E. Methods in Molecular Biology. Edited by LO, B. Totowa, N.J.:Humana Press, 2004. ISBN 1588290921. p. 227-242). Said bispecificantibodies may well use shorter linkers then those described above forscFv, i.e., having only one to three repeats of the basic motif of SEQID NO: 14 (see, e.g., HOLLIGER, P., et al. Diabodies: small bivalent andbispecific antibody fragments. PNAS 1993, vol. 90, no. 14, p.6444-6448). In another embodiment the multivalent binding member is atriabody, a minibody or tetrabody.

The invention also provides T-bodies comprising the antibodies disclosedherein. T-bodies are immunoglobulin T-cell receptors (cIgTCRs) whichcombine the antigen recognition of antibodies with the signal andeffector properties of the T-cell receptor complex. In such constructsthe antibody is preferably an antibody fragment such as a Fv, a Fab, ascFv or a scFv-Fc, most preferably a scFv. For further discussion of thegeneral design of T-bodies and their applications, see, e.g.,SCHIRRMANN, T. and Pecher, G. Handbook of Therapeutic Antibodies. Editedby DÜBEL, S. Weinheim: Wiley-VCH, 2009. ISBN 3527314539. p. 533-561.

The invention further provides a naïve (i.e., being not engineered forincreased affinity or potency) binding member against IL-1 beta having aMonovalent Potency (measured, e.g., as affinity (K_(D)) or biologicalpotency in cell-based assays (IC₅₀) in units of mol/1) at a certainMolecular Weight (in g/mol) after normalization to the Number of BindingSites per binding member, as determined by the equation

$K = \frac{{Monovalent}\mspace{14mu} {Potency}\mspace{14mu} ( {{mol}\text{/}l} )*{Molecular}\mspace{14mu} {Weight}\mspace{14mu} ( {g\text{/}{mol}} )}{{Number}\mspace{14mu} {of}\mspace{14mu} {Binding}\mspace{14mu} {Sites}}$

As described above, monovalent antibody fragments having potency valuesin the picomolar range are particular and not routinely obtained.Potency often correlates with the size of the binding member: highpotency in the picomolar range can be obtained by full-lengthimmunoglobulins, whereas very small antibody fragments such asnanobodies or minimal recognition units, or small non-antibody scaffoldssuch as affilins often show lower potency values, i.e., in the nanomolarrange. Seemingly, there is a minimum for said function K provided byscFv as described herein: the smaller the binding member, and the higherits monovalent potency or affinity, and the more binding sites permolecule, the smaller K. For example, for scFv as described herein, thelower limit of K equals about 50 ng/1 whereas the upper limit of Kequals about 12′500 ng/1; for the respective full-lengthimmunoglobulins, the lower limit equals about 150 ng/1 and the upper Klimit equals about 37′500 ng/1; for other binding members havingmolecular weights smaller than the scFv as described herein, the K valueis K>500′000 ng/l. In a preferred embodiment, the K value is about 50ng/l, 100 ng/l, 200 ng/l, 500 ng/l, 750 ng/l, 1′000 ng/l, 1′250 ng/l,1′500 ng/l, 1′750 ng/l, 2′000 ng/l, 2′250 ng/l, or 2′500 ng/l.

Nucleic Acids, Vectors, Host Cells and Method of Production

The antibodies described herein are encoded by a single nucleic acid orby two or more nucleic acids, for example each encoding at least onevariable region. Knowing the sequence of the antibody or of its parts,cDNAs encoding the polypeptide sequence can be generated by methods wellknown in the art, e.g. by gene synthesis. These cDNAs can be cloned bystandard cloning and mutagenesis techniques into a suitable vector suchas an expression vector or a cloning vector. Optionally, the variablelight chain is encoded by a separate nucleic acid than the variableheavy chain of the antibody. Further, additional sequences such as tags(e.g., a His-tag), constant domains for the production of a Fab or afull-length immunoglobulin, linkers, the coding sequence of a secondbinding specificity or another functional polypeptide such as an enzymeto generate a fusion construct or a bispecific molecule may be includedinto the genetic construct.

Based on the cloning strategy chosen, genetic constructs may generate anantibody having one or more additional residues at the N-terminal orC-terminal end. For example, an N-terminal methionine derived from thestart codon or an additional alanine may be present in an expressedpolypeptide, unless it has been clipped off post-translationally. It istherefore to be understood that the antibodies disclosed herein comprisethe disclosed sequences rather than consist of them.

In one embodiment, the invention provides a nucleic acid sequencecomprising at least 300 nucleobases, more preferably at least 350, 400,450, or 500 nucleobases and having at least 85%, more preferably atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID No.: 17. In a much preferred embodiment the nucleicacid sequence is SEQ ID No.: 17.

Additionally or alternatively, the invention provides a nucleic acidsequence comprising at least 300 nucleobases, more preferably at least350, 400, 450, or 500 nucleobases, which hybridizes with the nucleicacid of SEQ ID No.: 17 under high stringency conditions.

Basic protocols of standard cloning, mutagenesis and molecular biologytechniques are described in, e.g., Molecular Cloning, A LaboratoryManual (GREEN, M. and Sambrook, J. Molecular Cloning: a LaboratoryManual. 4th edition. Cold Spring Harbor Laboratory, 2012. ISBN1936113422).

Appropriate host cells for the expression of the genetic constructs canbe prokaryotic or eukaryotic. Suitable prokaryotic host cells aregram-negative or gram-positive and include species of the Escherichia,Erwinina, Enterobacter, Klebsiella, Pseudomonas or Bacillus families.Much preferred is Escherichia coli, in particular E. coli strains BL21(DE3) (LIFE TECHNOLOGIES™, cat. no. C6000-03) and ORIGAMI 2(DE3)(Novagen, cat. no 71345).

If post-translational modifications such as glycosylation orphosphorylation are desired, eukaryotic host cells are preferable. Forexample, eukaryotic microbes such as commonly used Saccharomycescerevisiae or Pichia pastoris strains may serve as host cells. Hostcells can also include plant or animal cells, in particular insect ormammalian cells. Suitable mammalian cells include, without being limitedto, Chinese Hamster Ovary Cells (CHO), Human Embryonic Kidney Cells(HEK), Human Umbilical Vein Endothelial Cells (HUVEC) or NSO myelomacells.

The antibody can be produced by expression in a suitable host cell. Forexample, the expression vectors described above are introduced into ahost cell by standard techniques such as electroporation or chemicaltransformation. The transformed cells are then cultivated underconditions adequate for recombinant protein expression, typically inappropriate nutritional media, optionally modified for inducingpromotors, selecting transformants, or amplifying encoding sequences ofinterest. The antibody is recovered from the culture and optionallypurified using standard techniques in the art. The yield of recombinantprotein may be improved by optimizing media and culture conditions suchas temperature or oxygen supply. In prokaryotes the antibody can beproduced in the periplasm, intracellularly as inclusion bodies or besecreted into the medium. Upon harvest, the protein can be purifiedusing methods well known in that art such as gel filtration, ionexchange chromatography, reversed phase chromatography, hydrophobicinteraction, mixed mode chromatography and/or affinity chromatography.

In one embodiment the antibody is produced in a cell-free system. Thistypically involves in vitro transcription followed by in vitrotranslation of nucleic acid product templates encoding the proteinsdescribed herein, e.g., plasmid DNA or PCR product templates. Forexample, crude lysates from growing cells are used, providing thenecessary enzymes as well as the cellular protein synthesis machinery.The necessary building blocks such as amino acids or nucleobases as wellas energy delivering molecules and others can be exogenously supplied.Cell-free expression systems can, for example, be based on lysed rabbitreticulocytes (e.g., Rabbit Reticulocyte Lysate System, Promega, cat.no. L4540), HeLa cells (e.g., 1-Step Human In Vitro Translation Kit,Thermo Scientific, cat. no. 88881), insect cells (e.g., EasyXpressInsect Kit II, Qiagen, cat. no. 32561), wheat germs (e.g., Wheat GermExtract, Promega, cat. no. L4380), or E. coli cells (e.g., PUREXPRESS®In Vitro Protein Synthesis Kit, NEB, cat. no. E6800S). Also, optimizedcell-free antibody expression systems for improved disulfide bondgeneration can be used for production. Commercially available kitsinclude insect cell lysates (e.g., EasyXpress Disulfide Insect Kit,Qiagen, cat. no. 32582) or E. coli cell lysates (e.g., EasyXpressDisulfide E. coli Kit, Qiagen, cat. no. 32572). Cell-free proteinsynthesis has, e.g., the advantage of being fast, achieving high productyields, allowing for easy modification of reaction conditions, forming alow degree of or even no byproducts. Cell-free protein synthesis mayinvolve biological and/or chemical steps which cannot be conducted inpurely biological or chemical production systems. For example,non-natural or chemically-modified amino acids can be incorporated intothe protein at desired positions. ScFv-toxin fusion proteins have beensuccessfully produced in cell-free systems (NICHOLLS, P. J., et al.Characterization of single-chain antibody (sFv)toxin fusion proteinsproduced in vitro in rabbit reticulocyte lysate. Journal of BiologicalChemistry 1993, vol. 268, pp. 5302-5308). Thus, in one embodiment amethod of producing the antibody described herein, the binding memberabove or the T-body above is provided comprising the steps of: (a)providing a cell-free system, (b) providing a nucleic acid producttemplate encoding the antibody described herein, the binding memberabove or the T-body above, (c) allowing for transcription andtranslation of said nucleic acid product template; (d) recovering, andoptionally (e) purifying said antibody, said binding member or saidT-body, respectively.

Additionally or alternatively, a method of producing the antibodydescribed herein comprises at least one step of chemical synthesis. Forexample, the method may be entirely chemical. In another embodiment, thecell-based or the cell-free production systems described above comprisesuch at least one step of chemical synthesis.

In a preferred embodiment the antibodies described herein are producedin a cell-based system using an expression vector for intracellularexpression in E. coli. Upon expression the polypeptide is generated asinclusion bodies within the cells which are separated from further cellparticles followed by solubilisation in a denaturing agent such asguanidine hydrochloride (GndHCl) and refolded by renaturation procedureswell known to the skilled person.

It is to be understood that the nucleic acids, vectors, host cells andmethod of production described above also apply to the binding members(insofar as they are a protein) and/or to T-bodies described herein.

Chemical and/or Biological Modifications

In one aspect the antibody of the instant invention is chemically and/orbiologically modified. Such modification may comprise, but is notlimited to, glycosylation, PEGylation, HESylation, Albumin fusiontechnology, PASylation, labelling with dyes and/or radioisotopes,conjugation with enzymes and/or toxins, phosphorylation, hydroxylationand/or sulfation. Likewise, any binding member, the nucleic acidsequence, the vector and/or the host cell described above can bemodified accordingly.

Chemical and/or biological modifications may be conducted to optimizepharmacodynamics or water solubility of the protein or to lower its sideeffects. For example, PEGylation, PASylation and/or HESylation may beapplied to slow down renal clearance and thereby increase plasmahalf-life time of the antibody. Additionally or alternatively, amodification may add a different functionality to the protein, e.g. atoxin to more efficiently combat cancer cells, or a detection moleculefor diagnostic purposes.

Glycosylation refers to a process that attaches carbohydrates toproteins. In biological systems, this process is performed enzymaticallywithin the cell as a form of co-translational and/or post-translationalmodification. A protein, here the antibody, can also be chemicallyglycosylated. Typically, but not limited to, glycosylation is (i)N-linked to a nitrogen of asparagine or arginine side-chains; (ii)O-linked to the hydroxy oxygen of serine, threonine, tyrosine,hydroxylysine, or hydroxyproline side-chains; (iii) involves theattachment of xylose, fucose, mannose, and N-acetylglucosamine to aphospho-serine; or (iv) in form of C-mannosylation wherein a mannosesugar is added to a tryptophan residue found in a specific recognitionsequence. Glycosylation patterns can, e.g., be controlled by choosingappropriate cell lines, culturing media, protein engineeringmanufacturing modes and process strategies (HOSSLER, P. Optimal andconsistent protein glycosylation in mammalian cell culture. Glycobiology2009, vol. 19, no. 9, p. 936-949).

Protein engineering to control or alter the glycosylation pattern mayinvolve the deletion and/or the addition of one or more glycosylationsites. The creation of glycosylation sites can conveniently beaccomplished by introducing the corresponding enzymatic recognitionsequence into the amino acid sequence of the antibody or by adding orsubstituting one or more of the above enumerated amino acid residues.

It may be desirable to PEGylate the antibody. PEGylation may alter thepharmacodynamic and pharmacokinetic properties of a protein.Polyethylene-glycol (PEG) of an appropriate molecular weight iscovalently attached to the protein backbone (see, e.g., PASUT, G. andVeronese, F. State of the art in PEGylation: the great versatilityachieved after forty years of research. Journal of Controlled Release2012, vol. 161, no. 2, p. 461-472). PEGylation may additionally reducethe immunogenicity by shielding the PEGylated protein from the immunesystem and/or alter its pharmacokinetics by, e.g. increasing the in vivostability of the antibody, protecting it from proteolytic degradation,extending its half-life time and by altering its biodistribution.

Similar effects may be achieved by PEG Mimetics, e.g., HESylating orPASylating the antibody. HESylation utilises hydroxyethyl starch (“HES”)derivatives, whereas during PASylation the antibody becomes linked toconformationally disordered polypeptide sequences composed of the aminoacids proline, alanine and serine. Said PEG Mimetics and relatedcompounds are, e.g., described in BINDER, U. and Skerra, A. Half-LifeExtension of Therapeutic Proteins via Genetic Fusion to Recombinant PEGMimetics, in Therapeutic Proteins: Strategies to Modulate Their PlasmaHalf-Lives. Edited by KONTERMANN, R., Weinheim, Germany: Wiley-VCH,2012. ISBN: 9783527328499. p. 63-81.

The antibody may include an epitope and in particular a salvage receptorbinding epitope. Such salvage receptor binding epitope typically refersto an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2,IgG3, or IgG4) and has the effect of increasing the in vivo half-life ofthe molecule.

Additionally or alternatively, the antibody is labelled with orconjugated to a second moiety which ascribes ancillary functionsfollowing target binding. Said second moiety may, e.g., have anadditional immunological effector function, be effective in drugtargeting or useful for detection. The second moiety can, e.g., bechemically linked or fused genetically to the antibody using knownmethods in the art.

Molecules which may serve as second moiety include, without beinglimited to, radionuclides, also called radioisotopes (e.g., ³⁵S ³²p,¹⁴C, ¹⁸F, ¹²⁵I); apoenzymes; enzymes (such as alkaline phosphatase,horseradish peroxidase, beta-galactosidase or angiogenin); co-factors;peptides (e.g., HIS-tags); proteins (incl. lectins); carbohydrates(incl. mannose-6-phosphate tag); fluorophores (including fluoresceinisothiocyanate (FITC); phycoerythrin; green/blue/red and otherfluorescent proteins; allophycocyanin (APC)); chromophores; vitamins(including biotin); chelators; antimetabolites (e.g., methotrexate),liposomes; toxins including cytotoxic drugs such as taxol, gramicidin Dor colchicine; or a radiotoxin.

A labelled antibody is particularly useful for in vitro and in vivodetection or diagnostic purposes. For example, an antibody labelled witha suitable radioisotope, enzyme, fluorophore or chromophore can bedetected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), or flow cytometry-based single cell analysis (e.g., FACSanalysis), respectively. Similarly, the nucleic acids and/or vectorsdisclosed herein can be used for detection or diagnostic purposes, e.g.using labelled fragments thereof as probes in hybridization assays.Labelling protocols may, e.g., be found in JOHNSON, I. and Spence, M. T.Z. Molecular Probes Handbook, A Guide to Fluorescent Probes and LabelingTechnologies. Life Technologies, 2010. ISBN: 0982927916.

Compositions

The antibody of the instant invention, any binding member, the nucleicacid sequences or the vector disclosed herein can be provided in acomposition which further comprises a suitable carrier, excipient ordiluent. Much preferred is a composition comprising an antibodydescribed herein.

Such composition can, e.g., be a diagnostic, a cosmetic or apharmaceutical composition. For therapeutic or cosmetic purposes, saidcomposition is a pharmaceutical composition comprising a pharmaceuticalcarrier, excipient or diluent, i.e. not being toxic at the dosages and aconcentration employed.

Suitable “carrier”, “excipients” or “diluents” include, without beinglimited to: (i) buffers such as phosphate, citrate, or other, organicacids; (ii) antioxidants such as ascorbic acid and tocopherol; (iii)preservatives such as 3-pentanol, hexamethonium chloride, benzalkoniumchloride, benzyl alcohol, alkyl paraben, catechol, or cyclohexanol; (iv)amino acids, such as e.g. histidine, arginine; (v) peptides, preferablyup to 10 residues such as polylysine; (vi) proteins, such as bovine orhuman serum albumin; (vii) hydrophilic polymers such aspolyvinylpyrrolidone; (viii) monosaccharides, disaccharides,polysaccharides and/or other carbohydrates including glucose, mannose,sucrose, mannitol, trehalose, sorbitol, aminodextran or polyamidoamines;(ix) chelating agents, e.g. EDTA; (x) salt-forming ions such as sodium;(xi) metal complexes (e.g. Zn-protein complexes); and/or (xii) ionic andnon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

Many of said exemplary compounds have different functions and may, e.g.,act as carrier and as diluent. It is also to be understood that thecomposition may comprise more than one of each carrier, diluent orexcipient.

The antibody, the binding member, the nucleic acid sequences or thevector may be provided on solid support materials such as beads andmicroparticles. Typically, the molecules are linked to such carrier viaa covalent bond (optionally involving a linker), noncovalently oradmixture. Said beads and microparticles can comprise, for example,starch, cellulose, polyacrylate, polylacetate polyglycolate,poly(lactide-co-glycolide), latex, or dextran.

Therapeutic Applications

The molecules described herein, in particular the antibody, bindingmember, nucleic acid or vector, are useful as a medicament. Typically,such medicament comprises a therapeutically effective amount of themolecules provided herein. Accordingly, said molecules can be used forthe production of a medicament useful in the treatment of IL-1beta-related disorders.

In one aspect, a method of treating an IL-1 beta-related disorder isprovided comprising the steps of administering a pharmaceuticallyeffective amount of the molecules described herein, in particular theantibody, to a subject in need thereof. In one embodiment, thepharmaceutical composition above (i.e., medicament) comprising suchpharmaceutically effective amount of the antibody is administered tosaid subject.

The term “treat” or “treatment” as used herein refers to theadministration of a pharmaceutically effective amount of the antibody,binding member, nucleic acid, vector or host cell of the instantinvention, to a subject in need thereof to prevent, cure, delay theonset and/or progression, reduce the severity of, stabilize, modulate,cure or ameliorate one or more symptoms of an IL-1 beta-relateddisorder. Typically, the antibody, binding member, nucleic acid, vectoror host cell is provided in a pharmaceutical composition including thosepreviously described herein.

A “therapeutically effective amount” refers to an amount which at thedosage regimen applied yields the desired therapeutic effect, i.e., toreach treatment goals as defined above. The dosage will depend onvarious factors including patient and clinical factors (e.g., age,weight, gender, clinical history of the patient, severity of thedisorder and/or response to the treatment), the nature of the disorderbeing treated, the particular composition to be administered, the routeof administration, and other factors.

The subject in need of such treatment can be a human or a non-humananimal, e.g., a mouse, rat, rabbit, monkey, dog, horse, cow, chicken,guinea pig or pig. Typically, the subject is diagnosed with an IL-1beta-related disorder or may acquire such a disorder.

Examples of IL-1 beta-related disorders, in which antagonist of IL-1beta have shown therapeutic effects include, without being limited to,proliferative diabetic retinopathy, gouty arthritis, Schnitzlersyndrome, systemic juvenile idiopathic arthritis, rheumatoid arthritis,acute gouty arthritis, chronic gouty arthritis, urticaria, vasculitis,type 1 diabetes, type 2 diabetes, ankylosing spondylitis, recurrentmultifocal osteomyelitis, relapsing polychondritis, cyropyrin-associatedperiodic syndrome (CAPS), Behçet's disease, familial mediterraneanfever, chronic obstructive pulmonary disease, polymyalgia rheumatic,NALP3-mutations, pyoderma gangrenosum, chronic idiopathic urticarial,osteoarthritis, wet age-related macular degeneration, dry eye syndrome,pustular psoriasis, synovitis-acne-pustulosis-hyperostosis-osteitissyndrome, macrophage activation syndrome, periodic fever, adenitis,pharyngitis, aphthous ulcer syndrome, adult-onset Still's disease,mevalonate kinase deficiency, atherosclerosis, TNF-receptor associatedperiodic syndrome (TRAPS), acne vulgaris and/or acne inversa.

The term “CAPS” or cryopyrin-associated periodic syndrome is to beunderstood to include each of familial cold autoinflammatory syndrome(FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisysteminflammatory disease, also known as chronic infantile neurological,cutaneous and articular (CINCA) syndrome.

The pharmaceutical composition may be applied by differentadministration routes. Administration can be conducted, for example, butnot limited to, parenterally, e.g., intramuscularly, subcutaneously,intravenously as a bolus or by continuous infusion, intraarticularly,intrasynovially, intracerebrally, intracerebrospinally, intrathecally,epidurally, or intraperitoneally; orally; rectally; locally,urogenitally; topically, e.g., to the skin or the eye; intravitreally;intravenously; intraocularly; oticly; intranasally; by inhalation;dermally such as intradermally, subcutaneously or transdermally;sublingually; buccally, for example. Preferred are the topical, rectal,local, intranasal, intravenous and/or intradermal routes ofadministration.

The antibody of the instant invention, the binding member, the nucleicacid sequences, the vector or host cell can be combined with one or morefurther therapeutically effective compound. Said compound may either becapable of disrupting signalling via the IL-1 receptor, or alternativelyinhibit one or more different targets such as, e.g., other mediators ofinflammatory responses. Such compound(s) can be administeredsimultaneously or sequentially.

For therapeutic applications, the antibody may also be radiolabelled orlinked to a toxin or linked to another effector function as describedabove.

Diagnostic Applications and/or Detection Purposes

The antibody of the instant invention may be used for detection ordiagnostic purposes in vivo and/or in vitro. For example, a wide rangeof immunoassays involving antibodies for detecting the expression inspecific cells or tissues are known to the skilled person. Likewise, anybinding member, the nucleic acid sequence, the vector and/or the hostcell described previously can be used accordingly as detailed in thissection.

For such applications the antibody, binding member, the nucleic acidsequence, the vector or the host cell disclosed herein may be eitherlabelled or unlabelled. E.g., an unlabelled antibody may be used anddetected by a secondary antibody recognizing an epitope on the antibodydescribed herein.

In another embodiment the antibody, binding member, nucleic acidsequence, vector and/or host cell is conjugated with one or moresubstances which can be recognized by a detector substance(s), e.g., theantibody being conjugated with biotin which can be detected bystreptavidin. Likewise, the nucleic acids and/or vectors disclosedherein can be used for detection or diagnostic purposes, e.g., by usinglabelled fragments thereof as probes in hybridization assays.

In certain embodiments, any of the molecules provided herein, inparticular the antibody, is useful for detecting the presence of IL-1beta, preferably including full-length IL-1 beta, fragments thereofand/or precursors thereof, in a sample, preferably biological sample.The term “detecting” encompasses quantitative and/or qualitativedetection. In certain embodiments a biological sample comprises a cellor tissue from human patients. Non limiting examples of biologicalsamples include blood, urine, cerebrospinal fluid, biopsy, lymph and/ornon-blood tissues.

In certain embodiments, the method comprises contacting the biologicalsample with an anti-IL-1 beta antibody as described herein underconditions permissive for binding of the antibody to IL-1 beta, ifpresent, and detecting whether a complex is formed between the antibodyand IL-1 beta. Such method may be an in vitro or in vivo method. In oneembodiment an anti-IL-1 beta antibody is used to select subjectseligible for therapy with the antibody described herein, e.g., whereIL-1 beta is a biomarker for selection of patients. Similarly, insteadof the antibody, such method may involve the use of the binding memberabove or a T-body described herein.

In another aspect, the antibody is used in cosmetic applications, e.g.,for improving the aesthetic appearance of skin.

In a further aspect, a kit is provided comprising the antibody, apackaged combination of reagents with instructions for performing thedetection or diagnostic assay. The reagents are typically provided inpredetermined amounts of dry powders, usually lyophilized, includingexcipients which after dissolution will provide a reagent solutionhaving the appropriate concentration. Other additives such asstabilizers and/or buffers may also be included. If the antibody islabelled with an enzyme, the kit will typically include the accordingsubstrates and cofactors. Likewise, any binding member, the nucleic acidsequence, the vector and/or the host cell described previously can beused accordingly as detailed in this section.

SEQUENCE LISTING

The sequences disclosed herein are:

SEQ ID No: 1-VH CDR1 FSLSSAANIA SEQ ID No: 2-VH CDR2 IIYDSASTYYASWAKGSEQ ID No: 3-VH CDR3 ERAIFSGDFVL SEQ ID No: 4-VL CDR1 QASQSIDNWLSSEQ ID No: 5-VL CDR2 RASTLAS SEQ ID No: 6-VL CDR3 QNTGGGVSIASEQ ID No: 7-VH EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVLWGQGTLVTVSSSEQ ID No: 8-VL EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFG QGTKLTVLGSEQ ID No: 9-linker GGGGSGGGGSGGGGSGGGGS SEQ ID No: 10-DLX2323EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSEQ ID No: 11-CDR variant of VH CDR1 FSLSXXAMASEQ ID No: 12-CDR variant of VH CDR2 IIXXSASTXYASWAKGSEQ ID No: 13-CDR variant of VH CDR3 EXXXXXXXXXXSEQ ID No: 14-CDR variant of VL CDR1 QASQSIXXXLSSEQ ID No: 15-CDR variant of VL CDR2 XASXLASSEQ ID No: 16-CDR variant of VL CDR3 QNXGXXXXIASEQ ID No: 17-DNA sequence of DLX2323GAAATTGTTATGACCCAGAGCCCGAGCACCCTGAGCGCAAGCGTTGGTGATCGTGTGATTATTACCTGTCAGGCAAGCCAGAGCATTGATAATTGGCTGAGCTGGTATCAGCAGAAACCGGGTAAAGCACCGAAACTGCTGATTTATCGTGCAAGCACCCTGGCAAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAGTGGTGCAGAATTTACCCTGACCATTAGCAGCCTGCAGCCGGATGATTTTGCAACCTATTATTGTCAGAATACCGGTGGTGGTGTTAGCATTGCATTTGGTCAGGGCACCAAACTGACCGTTCTGGGTGGTGGCGGTGGATCCGGTGGGGGTGGTAGCGGAGGTGGTGGTTCAGGCGGTGGTGGCAGCGAAGTTCAGCTGGTTGAAAGTGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTACCGCAAGCGGTTTTAGCCTGAGCAGCGCAGCAATGGCATGGGTTCGTCAGGCACCTGGTAAAGGTCTGGAATGGGTTGGTATTATCTATGATAGCGCAAGCACCTATTATGCAAGCTGGGCAAAAGGTCGTTTTACCATTAGCCGTGATACCAGTAAAAATACCGTTTACCTGCAGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCACGTGAACGTGCAATTTTCAGCGGTGATTTTGTTCTGTGGGGTCAGGGAACCCTGGTTACCGTTAGCAGCSEQ ID No.: 18-FR-L1 of FW1.4 EIVMTQSPSTLSASVGDRVIITCSEQ ID No.: 19-FR-L2 of FW1.4 WYQQKPGKAPKLLIYSEQ ID No.: 20-FR-L3 of FW1.4 GVPSRFSGSGSGAEFTLTISSLQPDDFATYYCSEQ ID No.: 21-FR-L4 of FW1.4 FGQGTKLTVLG SEQ ID No.: 22-FR-H1 of rFW1.4EVQLVESGGGLVQPGGSLRLSCTASG SEQ ID No.: 23-FR-H2 of rFW1.4 WVRQAPGKGLEWVGSEQ ID No.: 24-FR-H3 of rFW1.4 RFTISRDTSKNTVYLQMNSLRAEDTAVYYCARSEQ ID No.: 25-FR-H4 of rFW1.4 WGQGTLVTVSSSEQ ID No.: 26-FR-H1 of rFW1.4(V2) EVQLVESGGGLVQPGGSLRLSCTVSGSEQ ID No.: 27-FR-H2 of rFW1.4(V2) WVRQAPGKGLEWVGSEQ ID No.: 28-FR-H3 of rFW1.4(V2) RFTISKDTSKNTVYLQMNSLRAEDTAVYYCARSEQ ID No.: 29-FR-H4 of rFW1.4(V2) WGQGTLVTVSSSEQ ID No.: 30-FR-H1 of rFW1.4-SST EVQLVESGGGSVQPGGSLRLSCTASGSEQ ID No.: 31-FR-H2 of rFW1.4-SST WVRQAPGKGLEWVGSEQ ID No.: 32-FR-H3 of rFW1.4-SST RFTISRDTSKNTVYLQMNSLRAEDTASYYCARSEQ ID No.: 33-FR-H4 of rFW1.4-SST WGQGTTVTVSSSEQ ID No.: 34-CDR-L1 D32XEIVMTQSPSTLSASVGDRVIITCQASQSITWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 35-CDR-L1_N33XEIVMTQSPSTLSASVGDRVIITCQASQSIDWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 36-CDR-L1_W40XEIVMTQSPSTLSASVGDRVIITCQASQSIDNXLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of glutamic acid(E), phenylalanine (F), glycine (G), methionine (M), asparagine (N),glutamine (Q), serine (S), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 37-CDR-L2_R58XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYXASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 38-CDR-L2_T69XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASXLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 39-CDR-L3_T109XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNXGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), isoleucine (I), asparagine (N), serine (S), threonine (T)and valine (V).

SEQ ID No.: 40-CDR-L3_G111XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGXGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),glycine (G), proline (P) and serine (S).

SEQ ID No.: 41-CDR-L3_G112XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGXVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 42- CDR-L3_V135XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGXSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 43-CDR-L3_S136XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVXIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M),asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 44-CDR-H1_S33XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSXAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 45-CDR-H1_A39XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSXAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 46-CDR-H2_Y59XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIXDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), glycine (G), methionine (M) and tyrosine (Y).

SEQ ID No.: 47-CDR-H2_D60XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYXSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of aspartic acid(D), asparagine (N) and proline (P).

SEQ ID No.: 48-CDR-H2_Y69XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTXYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),aspartic acid (D), glutamic acid (E), glycine (G), phenylalanine (F),histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M),proline (P), asparagine (N), serine (S), threonine (T), tryptophan (W)and tyrosine (Y).

SEQ ID No.: 49-CDR-H3_R110XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAIVIAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAREXAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 50- CDR-H3_A111XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERXIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), phenylalanine (F), glycine (G),histidine (H), isoleucine (I), lysine (K), methionine (M), asparagine(N), proline (P), glutamine (Q), arginine (R), serine (S), threonine(T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 51-CDR-H3_I112XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAANIAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAXFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), phenylalanine (F), histidine (H), isoleucine (I), leucine(L), methionine (M), asparagine (N), glutamine (Q), serine (S),threonine (T), valine (V) and tyrosine (Y).

SEQ ID No.: 52-CDR-H3_F113XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIXSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of phenylalanine (F)and isoleucine (I).

SEQ ID No.: 53-CDR-H3_S114XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFXGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), glutamic acid (E), glycine (G), serine (S), threonine (T)and valine (V).

SEQ ID No.: 54-CDR-H3_G115XEIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSXDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),glycine (G), methionine (M) and asparagine (N).

CDR-H3_D135X SEQ ID No.: 55EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGXFVLWGQGT LVTVSS

Preferably, X is selected from the group consisting of alanine (A),aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N),serine (S) and threonine (T).

CDR-H3_F136X SEQ ID No.: 56EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAIVIAWVRQAPGKGLEWVGITYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDXVLWGQ GTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine(I), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine(S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

CDR-H3_V137X SEQ ID No.: 57EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFXLWGQGT LVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

CDR-H3_L138X SEQ ID No.: 58EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVWGQGTL VTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

DLX2464 SEQ ID No.: 59EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDMAGWGQGTLVTVSS DLX2465 SEQ ID No.: 60EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMDLWGQGTLVTVSS DLX2466 SEQ ID No.: 61EIVMTQSPSTLSASVGDRVIITCQASQSIGKYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSDAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMA GWGQGTLVTVSSDLX2467 SEQ ID No.: 62EIVMTQSPSTLSASVGDRVIITCQASQSIHNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGSSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSRAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERMIFSGDFV LWGQGTLVTVSSDLX2468 SEQ ID No.: 63EIVMTQSPSTLSASVGDRVIITCQASQSIGNYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGTSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMV LWGQGTLVTVSSDLX2475 SEQ ID No.: 64EIVMTQSPSTLSASVGDRVIITCQASQSIDKWLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVHIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSYAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFKLWGQGTLVTVSS DLX2476 SEQ ID No.: 65EIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERDIFSGDFV GWGQGTLVTVSSDLX2480 SEQ ID No.: 66EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDFV LWGQGTLVTVSSDLX2543 SEQ ID No.: 67EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS DLX2529 SEQ ID No.: 68EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKWYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFV LWGQGTLVTVSSDLX2547 SEQ ID No.: 69ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFV LWGQGTLVTVSSDLX2528 SEQ ID No.: 70EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS DLX2585 SEQ ID No.: 71EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS DLX2545 SEQ ID No.: 72EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISRDTSKNTLYLQMNSLRAEDTAVYFCARERNIFSGDMV LWGQGTTVTVSSDLX2531 SEQ ID No.: 73EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS DLX2586 SEQ ID No.: 74EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYFCARERQIFSGDMD GWGQGTLVTVSSDLX2530 SEQ ID No.: 75EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS DLX2548 SEQ ID No.: 76EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKWYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMD GWGQGTTVTVSSDLX2676 SEQ ID No.: 77EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS DLX2677 SEQ ID No.: 78EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS DLX2678 SEQ ID No.: 79EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMD GWGQGTTVTVSSDLX2679 SEQ ID No.: 80EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS DLX2680 SEQ ID No.: 81EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKWYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMA LWGQGTTVTVSSDLX2681 SEQ ID No.: 82EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFA LWGQGTLVTVSSDLX2682 SEQ ID No.: 83EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDG WGQGTTVTVSSDLX2683 SEQ ID No.: 84EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFD YWGQGTLVTVSSDLX2684 SEQ ID No.: 85EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVTVSS DLX2685 SEQ ID No.: 86EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS DLX2686 SEQ ID No.: 87EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMD GWGQGTTVTVSSDLX2687 SEQ ID No.: 88EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS DLX2689 SEQ ID No.: 89ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVTVSS DLX2690 SEQ ID No.: 90ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMD GWGQGTTVTVSSDLX2691 SEQ ID No.: 91ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTVSS DLX2692 SEQ ID No.: 92ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMA LWGQGTTVTVSSDLX2693 SEQ ID No.: 93ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFA LWGQGTLVTVSSDLX2694 SEQ ID No.: 94ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDG WGQGTTVTVSSDLX2695 SEQ ID No.: 95ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFD YWGQGTLVTVSSVL CDR-L1_D32X SEQ ID No.: 96EIVMTQSPSTLSASVGDRVIITCQASQSIWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

VL CDR-L1_N33X SEQ ID No.: 97EIVMTQSPSTLSASVGDRVIITCQASQSIDXWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

VL CDR-L1_W40X SEQ ID No.: 98EIVMTQSPSTLSASVGDRVIITCQASQSIDNXLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of glutamic acid(E), phenylalanine (F), glycine (G), methionine (M), asparagine (N),glutamine (Q), serine (S), tryptophan (W) and tyrosine (Y).

VL CDR-L2_R58X SEQ ID No.: 99EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYXASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), tryptophan (W) and tyrosine (Y).

VL CDR-L2_T69X SEQ ID No.: 100EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASXLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

VL CDR-L3_T109X SEQ ID No.: 101EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNXGGGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), isoleucine (I), asparagine (N), serine (S), threonine (T)and valine (V).

VL CDR-L3_G111X SEQ ID No.: 102EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGXGVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),glycine (G), proline (P) and serine (S).

VL CDR-L3_G112X SEQ ID No.: 103EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGXVS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

VL CDR-L3_V135X SEQ ID No.: 104EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGXS IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

VL CDR-L3_S136X SEQ ID No.: 105EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVX IAFGQGTKLTVLG

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M),asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

VH CDR-H1_S33X SEQ ID No.: 106EVQLVESGGGLVQPGGSLRLSCTASGFSLSXAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCA RERAIFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 107-VH CDR-H1_A39XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSXAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 108-VH CDR-H2_Y59XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIXDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), glycine (G), methionine (M) and tyrosine (Y).

SEQ ID No.: 109-VH CDR-H2_D60XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYXSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of aspartic acid(D), asparagine (N) and proline (P).

SEQ ID No.: 110-VH CDR-H2_Y69XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTXYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),aspartic acid (D), glutamic acid (E), glycine (G), phenylalanine (F),histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M),proline (P), asparagine (N), serine (S), threonine (T), tryptophan (W)and tyrosine (Y).

SEQ ID No.: 111-VH CDR-H3_R110XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAREXA IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S),threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 112-VH CDR-H3_A111XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERX IFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), phenylalanine (F), glycine (G),histidine (H), isoleucine (I), lysine (K), methionine (M), asparagine(N), proline (P), glutamine (Q), arginine (R), serine (S), threonine(T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 113-VH CDR-H3_I1112XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAANIAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARER AXFSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), phenylalanine (F), histidine (H), isoleucine (I), leucine(L), methionine (M), asparagine (N), glutamine (Q), serine (S),threonine (T), valine (V) and tyrosine (Y).

SEQ ID No.: 114-VH CDR-H3_F113XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IXSGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of phenylalanine (F)and isoleucine (I).

SEQ ID No.: 115-VH CDR-H3_S114XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFXGDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), glutamic acid (E), glycine (G), serine (S), threonine (T)and valine (V).

SEQ ID No.: 116-VH CDR-H3_G115XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSXDFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),glycine (G), methionine (M) and asparagine (N).

SEQ ID No.: 117-VH CDR-H3_D135XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGXFVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N),serine (S) and threonine (T).

SEQ ID No.: 118-VH CDR-H3_F136XEVQLVESGGGLVQPGGSLRLSCTASGFSLS SAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARER AIFSGDXVLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine(I), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine(S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

SEQ ID No.: 119-VH CDR-H3_V137XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFXLWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 120-VH CDR-H3_L138XEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERA IFSGDFVWGQGTLVTVSS

Preferably, X is selected from the group consisting of alanine (A),cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F),glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L),methionine (M), asparagine (N), proline (P), glutamine (Q), arginine(R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine(Y).

SEQ ID No.: 121-VH DLX2464EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDMAGWGQGTLV TVSSSEQ ID No.: 122-VH DLX2465EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMDLWGQGTLV TVSSSEQ ID No.: 123-VL DLX2466EIVMTQSPSTLSASVGDRVIITCQASQSIGKYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGSEQ ID No.: 124-VH DLX2466EVQLVESGGGLVQPGGSLRLSCTASGFSLSSDAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMAGWGQGTLV TVSSSEQ ID No.: 125-VL DLX2467EIVMTQSPSTLSASVGDRVIITCQASQSIHNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGSSIAFGQGTKLTVLGSEQ ID No.: 126-VH DLX2467EVQLVESGGGLVQPGGSLRLSCTASGFSLSRAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERMIFSGDFVLWGQGTLVT VSSSEQ ID No.: 127-VL DLX2468EIVMTQSPSTLSASVGDRVIITCQASQSIGNYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNAGGGTSIAFGQGTKLTVLGSEQ ID No.: 128-VH DLX2468EVQLVESGGGLVQPGGSLRLSCTASGFSLSSSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERNIFSGDMVLWGQGTLVT VSSSEQ ID No.: 129-VL DLX2475EIVMTQSPSTLSASVGDRVIITCQASQSIDKWLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVHIAFGQGTKLTVLGSEQ ID No.: 130-VH DLX2475EVQLVESGGGLVQPGGSLRLSCTASGFSLSSYAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFKLWGQGTLVT VSSSEQ ID No.: 131-VL DLX2476EIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 132-VH DLX2476EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERDIFSGDFVGWGQGTLVT VSSSEQ ID No.: 133-VL DLX2480EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLTVLGSEQ ID No.: 134-VH DLX2480EVQLVESGGGLVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERQIFSGDFVLWGQGTLVT VSSSEQ ID No.: 135-VL DLX2543EIVMTQSPSTLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNAGGGVSIAFGQGTKLTVLGSEQ ID No.: 136-VL DLX2529EIVMTQSPSTLSASVGDRVIITCRASQSIGNWLSWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGAEFTLTISSLQPEDFATYYCQNTGGGINIAFGQGTKLTVLGSEQ ID No.: 137-VL DLX2547ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGINIAFGQGTKLEIKRSEQ ID No.: 138-VH DLX2547EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVT VSSSEQ ID No.: 139-VL DLX2528EIVMTQSPSTLSASVGDRVTITCQASQSIGNWLAWYQQKPGKAPKLLIYQASNLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQNAGGATTIAFGQGTKLTVLGSEQ ID No.: 140-VH DLX2528EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVT VSSSEQ ID No.: 141-VL DLX2585EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 142-VH DLX2585EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFDYWGQGTLVTV SSSEQ ID No.: 143-VL DLX2545EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 144-VH DLX2545EVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISRDTSKNTLYLQMNSLRAEDTAVYFCARERNIFSGDMVLWGQGTTVTV SSSEQ ID No.: 145-VL DLX2531EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 146-VH DLX2531EVQLVESGGGNVQPGGSLRLSCTASGFSLSNSAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCARERAIFSGDFALWGQGTLVT VSSSEQ ID No.: 147-VL DLX2586EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 148-VH DLX2586EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTLVT VSSSEQ ID No.: 149-VL DLX2530EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 150-VH DLX2530EVQLVESGGGNVQPGGSLRLSCTASGFSLSDAAMAWVRQAPGKGLEWVGIIYDSASTFYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCARERNIFSGDMALWGQGTTVT VSSSEQ ID No.: 151-VL DLX2548EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGSEQ ID No.: 152-VH DLX2548EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIYDSASTYYASWAKGRFTISKDTSKNTLYLQMNSLRAEDTAVYFCARERQIFSGDMDGWGQGTTVTV SSSEQ ID No.: 153-VL DLX2544ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAFGQGTKVEIKRSEQ ID No.: 154-DLX2544ADIVMTQSPSTLSASVGDRVTITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNAGGGINIAFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSS SEQ ID No.: 155-CDR-H1 of DLX2531 FSLSNSAMASEQ ID No.: 156-CDR-H2 of DLX2531 IIYDSASTYYASWAKGSEQ ID No.: 157-CDR-H3 of DLX2531 ERAIFSGDFALSEQ ID No.: 158-CDR-L1 of DLX2531 QASQSIDNWLSSEQ ID No.: 159-CDR-L2 of DLX2531 RASTLASSEQ ID No.: 160-CDR-L3 of DLX2531 QNTGGGVSIASEQ ID No.: 161-CDR-L1 of DLX2681 RASQSIGNWLSSEQ ID No.: 162-CDR-L2 of DLX2681 RASNLASSEQ ID No.: 163-CDR-L3 of DLX2681 QNTGGGINIA

EXAMPLES Example 1—Identification of rhIL-1 Beta Neutralizing scFv

Immunization of rabbits: Rabbits were immunized with recombinant humanIL-1 beta protein (Peprotech, USA, cat. no. 200-01B). Lymph node andspleen cells were isolated after the final boost and the cells werecryopreserved.

Flow cytometry sorting of rabbit B cells and culturing: IL-1beta-specific memory B cells were sorted as single cells into 96-wellmicroplates using FACSAria III (BD Biosciences). Single B cell cloneswere cultured in the presence of feeder cells.

Screening of B cell clones: Cell culture supernatants were analyzed byELISA for the presence of anti-IL-1 beta-specific IgGs. Briefly, rhIL-1beta (Peprotech, cat. no. 200-01B) was coated at a concentration of 2mcg/ml overnight at 4° C. on Maxisorp 96-well microplates in PBS. Afterblocking with 5% non-fat dry milk, cell culture supernatants were added.IL-1 beta-specific IgGs were detected by anti-rabbit IgG-HRP (SouthernBiotech, cat. no. 4050-05). The ELISA was developed with BM Blue PODsubstrate (Roche Applied Science). B cell clones specific for rhIL-1beta were further analyzed for their neutralization capacity in a humanfibroblast assay.

Sequencing of IL-1 beta-neutralizing IgGs: all rabbit B cell clonesproducing neutralizing anti-IL-1 beta antibodies were subjected to mRNAisolation using the RNeasy Mini Kit (Qiagen Germany, cat. no. 74106).mRNA was used as template for reverse transcription according to themanufacture's protocol (OneStep RT-PCR kit, Qiagen Germany, cat. no.210212). Subsequently, PCR reactions using oligonucleotides tospecifically amplify rabbit IgG heavy and light chain encoding sequenceswere carried out (Biometra Thermocycler T3). Heavy and light chain PCRfragments were independently sequenced (ABI, Sanger 3730xl; MicrosynthAG, Balgach, Switzerland), and obtained DNA sequences were translatedinto protein sequences using EMBOSS Transeq and aligned using CLUSTALW2.

Construction of anti-IL-1 beta scFv genes, and scFv protein expression:rabbit IgG CDR regions of the light and the heavy chains as definedabove were identified and grafted into the human light and heavy chainacceptor frameworks comprising SEQ ID Nos.: 18-21 and 22-25,respectively. Bacterial expression vectors were generated encoding scFvproteins with the N-terminal variable light chain linked by the sequenceSEQ ID No: 9 to the C-terminal variable heavy chain. ScFv proteins wereexpressed in E. coli BL21 (DE3); Novagen, USA, cat. no. 69450-3) asinclusion bodies, which were purified, solubilized and the proteins wererefolded. The refolded scFvs were purified by standard size exclusionchromatography and monomeric peak fractions were collected. PurifiedscFvs were analyzed for IL-1 beta binding by ELISA. ScFv that were foundto specifically bind rhIL-1 beta were tested for IL-1 betaneutralization in a human fibroblast assay. By this procedure, the scFvDLX2323 and other anti-IL-1 beta scFvs were identified as potentinhibitors of IL-1 beta.

Example 2—Recognition of Human IL-1 Beta

Firstly, the specific recognition of rhIL-1 beta by DLX2323 wasconfirmed by ELISA (FIG. 1). Briefly, rhIL-1 beta (Peprotech, cat. no.200-01B) was coated at a concentration of 2 mcg/ml overnight at 4° C. onMaxisorp 96-well microplates in PBS. After blocking with 5% non-fat drymilk, increasing concentrations of scFvs (10 to 300 ng/ml) were added,and scFvs were detected by Protein L-HRP (Sigma-Aldrich, cat. no.P3226). The ELISA was developed with BM Blue POD substrate (RocheApplied Science). As a negative control, scFv of irrelevant specificitywas used. This result shows that DLX2323 specifically binds to rhIL-1beta.

To confirm that DLX2323 and the control scFv were recognized by ProteinL-HRP and, thus, a lack of signal for rhIL-1 beta binding in the ELISAabove was not due to a detection problem, another experiment wasconducted. The scFvs were directly coated on the plate and detected byProtein L-HRP as described above. All scFvs were coated at aconcentration of 2 mcg/ml in PBS. This ELISA experiment showed thatDLX2323 and the control scFv are recognized by the detection agentProtein L-HRP.

In another ELISA (FIG. 2), the recognition of human natural IL-1 beta byDLX2323 was confirmed. As commonly known, expression of human proteinsin cells other than human cells might cause changes, e.g., inpost-translational modifications and/or conformation. This might lead toa differential recognition of recombinant and natural proteins byantibodies. Natural human IL-1 beta was secreted by THP-1 cells (DSMZGermany, cat. no. ACC 16) after stimulation with 10 ng/ml of PMA(Sigma-Aldrich, cat. no. P1585), 1 mg/ml of LPS (Sigma-Aldrich, cat. no.L4391) and 2 mM of ATP (Sigma-Aldrich, cat. no. A6559-25UMO). Cellsupernatants were harvested and secreted human natural IL-1 beta wasquantified using the Human IL-1 beta/IL-1F2 ELISA DuoSet (R&D Systems,cat. no. DY201). DLX2323 was coated on 96-well microplates (Maxisorp,Nunc) at a coating density of 5 mcg/ml in DPBS (pH 7.4). Human IL-1 betaeither as recombinantly expressed version (Peprotech, cat. no. 200-01B)or as natively secreted version were applied at final concentrationsranging from 0.5 to 4 ng/ml. Bound IL-1 beta was detected by abiotinylated goat anti-hIL-1 beta antibody (R&D Systems, cat. no. DY201)and Streptavidin-HRP (BD Pharmingen, cat. no. 554060). The ELISA wasdeveloped using BM Blue POD substrate (Roche Applied Science). Forquantification purposes the absorbance was measured at 450 nm using aVersaMax microplate reader (Molecular Devices, USA). The result (seeFIG. 2) shows that both, recombinant and natural human IL-1 beta arerecognized by DLX2323 at comparable levels.

Example 3—Neutralization of rhIL-1 Beta Biological Activity

Antibodies and scFvs were tested for their IL-1 beta neutralizationcapacity in a human dermal fibroblast assay (NHDF-Neo, cat. no. CC-2509,Lonza Walkersville USA). Activation of such fibroblasts with IL-1 betaleads to specific IL-6 release which is quantified by ELISA. Inhibitionof IL-1 beta by specific antibodies decreases the amount of IL-6released from such fibroblasts. The inhibitory potency of the anti-IL-1beta antibody is quantified by measuring the half-maximal reduction(IC₅₀) of IL-1 beta-induced IL-6 release. Human dermal fibroblasts wereseeded in 96-well microplates at 5′000 cells/well 16-20 hours prior toaddition of IL-1 beta. The fibroblasts were cultured in fibroblast basalmedium (FBM; Lonza, cat. no. CC-3131) with supplements (hFGF-B, Insulin,FBS, GA-1000) as described by the cell supplier (Lonza Walkersville USA:CLONETICS™ Dermal Fibroblast Cell Systems). FBM then was removed andcells were washed once with Dulbecco's Modified Eagle Medium (DMEM;Gibco, Life Technologies, cat. no. 11880) to remove growth factors.Cells were then incubated for 7 hours in DMEM media. Antibodies or scFvsand rhIL-1 beta were pre-incubated in DMEM for 1 hour at 37° C. Themixture was added to the cells at a final concentration of 10 pg/ml ofIL-1 beta. As negative control, 10 pg/ml of IL-1 beta was added to cellswithout any anti-IL-1 beta antibody. As positive control, a mousemonoclonal antibody against IL-1 beta was applied (R&D systems, USA,cat. no. MAB201). The cells were incubated with the IL-1 beta/anti-IL-1beta antibody mixture for 18-24 hours, and cell culture supernatantswere analyzed for IL-6 release using the Human IL-6 DuoSet ELISA Kitaccording to the manufacturer's instructions (R&D Systems, USA, cat. no.DY206).

The IC₅₀ of DLX2323 was determined to be 3 pM±1.05 in eight independentassays.

Example 4—Comparison of Neutralization Potency with CommerciallyAvailable IL-1 Beta Inhibitors

DLX2323 was identified as an IL-1 beta neutralizing scFv. The biologicalpotency of DLX2323 and other inhibitors was assessed in the human dermalfibroblast assay as described in Example 3. Recombinant human IL-1 betawas pre-incubated with increasing concentrations of the scFv DLX2323,the anti-human IL-1 beta monoclonal IgG antibody MAB201, the IL-1 betareceptor antagonist (rhIL-1ra) (R&D systems, cat. no. 280-RA-010/CF) orthe FDA approved, marketed canakinumab IgG (Novartis, ILARIS®) prior toaddition to the wells. Two independent experiments were performed: FIG.3A depicts the comparison of DLX2323 with MAB201, whereas FIG. 3B showsthe comparison of DLX2323 with rhIL-1ra and canakinumab. The followingIC₅₀ values were determined: MAB201: 2-3 pM; DLX2323: 2-4 pM; rhIL-1ra:40 pM and Canakinumab: 90 pM. In conclusion, the monovalent monomericscFv DLX2323 is almost as potent as the bivalent monoclonal mouseantibody MAB201. It shows distinctly higher potency in neutralizinghuman IL-1 beta than the marketed inhibitor Canakinumab and therhIL-1ra. Furthermore, DLX2323 could fully block IL-1 beta-induced IL-6release.

Example 5—Solubility

DLX2323 was stored in PBS buffer pH 7.2 (Phosphate Buffered Saline 1×,Gibco, LIFE TECHNOLOGIES, cat. no. 20012). To determine its maximumsolubility, DLX2323 was concentrated using Vivaspin 20 centrifugeconcentrators (Sartorius Stedim Biotech, cat. no. VS2001) at roomtemperature. The concentration process was stopped at 71 mg/ml due tothe high viscosity of the sample. The obtained DLX2323 protein solutionwas viscous, clear, and no precipitates were observed by visualinspection.

Example 6—Stability

Regarding the stability of scFvs, two different processes can beobserved that contribute to their instability. Firstly, the scFv couldbe prone to dimerization, often followed by oligomerization andeventually aggregation. Secondly, scFv degradation, leading to smallerfragments, can occur over time. To judge whether DLX2323 is stable, HPLC(Dionex, Summit system) size exclusion chromatography (Tosoh, TSKgelG2000SWxl, cat. no. 08540) was deployed to determine the percentage ofmonomeric, non-degraded scFv protein at certain time points at differentprotein concentrations and temperatures (e.g., 4° C., RT and 37° C.).The percentage of monomer was measured at the starting point of thestudy (TO) and after one month for 1 mg/ml of DLX2323, and, in anotherexperiment, after two weeks for a 50 mg/ml solution of DLX2323. Theprotein was formulated in PBS, pH 7.2. The results of the stabilitystudy are listed in tables 1 and 2.

TABLE 1 monomer content after 1 month Observation DLX2323, 1 mg/ml, T097% DLX2323, 1 mg/ml, stored at RT 98% DLX2323, 1 mg/ml, stored at 37°C. 93% Very low levels of degradation

TABLE 2 monomer content after 2 weeks Observations DLX2323, 50 mg/ml, T097% DLX2323, 50 mg/ml, stored at 4° C. 78% dimerization DLX2323, 50mg/ml, stored at RT 76% oligomerization DLX2323, 50 mg/ml, stored at 37°C. 43% oligomerization

DLX2323's thermal stability was assessed by differential scanningfluorimetry (DSF). For this measurement a real-time PCR device (Corbett,Rotor-Gene) heated DLX2323 in a temperature gradient from 30° C. to 95°C. (raising in 1° C. steps, waiting 5 seconds per step). The proteinsample contained 0.5 mg/ml of DLX2323 and 20× SYPRO® Orange(Sigma-Aldrich, cat. no. 55692, 5000×) in PBS. As soon as the proteinstarted melting, Sypro Orange turned fluorescent. This fluorescence wasonline measured (excitation wavelength of 470 nm; emission wavelength of555 nm) during the gradient run. Using Rotor-Gene 6000 Series Software1.7 the midpoint melting temperature (Tm) of DLX2323 was calculated tobe 74° C.

Example 7—Cross-Reactivity

Cross-reactivity of DLX2323 to IL-1 beta homologs of other species thanhuman beings was assessed in ELISA. Binding to the recombinantlyexpressed IL-1 beta proteins of the following species was investigated:cynomolgus (Sino Biological Inc., USA, cat. no. 90010-CNAE), rhesusmacaque (R&D Systems, USA, cat. no. 1318-RL/CF), swine (KingfisherBiotech, USA, cat. no. RP0297S-025), canine (Kingfisher Biotech, USA,cat. no. RP0085D-025), guinea pig (Kingfisher Biotech, cat. no.RP0343GP-025), rat (Peprotech, cat. no. 400-01B) and mouse (BioLegend,cat. no. 575102). Binding of DLX2323 was compared to ELISA-positivecontrol antibodies (R&D Systems, USA, goat anti-human IL-1 betapolyclonal IgG, cat. no. AB-201-NA; BioLegend, Inc., USA, biotinanti-mouse/rat IL-1 beta antibody, cat. no. 503505). Briefly, proteinswere coated at a concentration of 2 mcg/ml over night at 4° C. onMaxisorp 96-well microplates in PBS. After blocking with 5% non-fat drymilk, increasing concentrations (0.1 mcg/ml, 0.3 mcg/ml and 1.0 mcg/ml)of DLX2323 were added to the wells. Successful coating of every proteinwas separately confirmed exploiting IL-1 beta-specific controlantibodies. Whereas DLX2323 was detected by Protein L-HRP(Sigma-Aldrich, USA, cat. no. P3226), the control antibodies weredetected by either Streptavidin-HRP (BD Pharmingen, USA, cat. no.554060) or other eligible secondary antibodies labelled with HRP. TheELISA was developed with BM Blue POD substrate (Roche Applied Science)and the absorbance was measured at 450 nm. DLX2323 recognized fourspecies orthologs of IL-1 beta, namely human, cynomolgus, rhesus macaqueand rat IL-1 beta. No cross-reactivity could be observed for porcine,guinea pig, canine and mouse IL-1 beta.

Besides the cross-reactivity of DLX2323 to IL-1 beta homologs of otherspecies than human beings, the recognition pattern of DLX2323 regardingvarious human IL-1 family members and other cytokines was measured:rhIL-1ra (R&D systems, USA, cat. no. 280-RA-010/CF), rhIL-1 alpha(PeproTech, cat. no. 200-01A), rhIL-18 (BioVision, cat. no. 4179-25),rhIL-33 (Peprotech, cat. no. 200-33), IL-36ra (R&D Systems, cat. no.1275-IL/CF), rhTNF alpha (Peprotech, Hamburg, Germany cat. no. 300-01A)and rhIL-6 (Peprotech, cat. no. 200-06). In the applied ELISA assay thefollowing antibodies served as positive controls: biotin anti-humanIL-1ra (BioLegend, cat. no. 509501), biotin anti-human IL-1 alpha(BioLegend, cat. no. 515703), anti-human IL-18 polyclonal antibody(BioVision, cat. no. 5179-100), biotin anti-human IL-33 antibody(Peprotech, cat. no. 500-P261Bt), anti-human TNF alpha scFv DLX105(Delenex propriety antibody described in WO 2006/131013 A), biotinanti-human IL-6 (R&D systems, DY206, cat. no. 840114), anti-humanIL-36ra (R&D systems, cat. no. AF1275). The ELISA was carried outessentially as described above. No cross-reactivities of DLX2323 for anyof these tested human IL-1 family members and cytokines could bedetected.

Example 8—In Vivo Efficacy

In this example, the in vivo inhibition of human IL-1 beta activity byDLX2323 is demonstrated. Human IL-1 beta can bind and activate the mouseIL-1 receptor thereby inducing an inflammatory response in the mouse invivo. The inflammation leads to elevated levels of cytokines in theserum including mouse IL-6 (mIL-6). Recombinant human IL-1 beta(Peprotech, cat. no. 200-01B) was administered subcutaneously at a doseof 1.5 mcg/kg body weight to 8 weeks old male BALB/c mice (CharlesRiver, Germany). After 2 hours mIL-6 levels were significantly elevatedin serum. To test the neutralization capacity of DLX2323 in vivo, it wasintraperitoneally (i.p.) injected two hours prior to the IL-1 betadosing. One group of mice was injected with a 5 mg/kg dose of DLX2323,the second group was injected with a 15 mg/kg dose of DLX2323. Negativecontrol groups were treated with either PBS i.p. or scFvs of irrelevantspecificity. A fifth group of mice was intravenously injected with 10mg/kg of canakinumab (Novartis, ILARIS®) as a positive control. Twohours after the rhIL-1 beta application blood samples were taken andserum levels of mIL-6 were measured using the Mouse IL-6 DuoSet ELISAkit according to the manufacturer's instructions (R&D Systems, cat. no.DY406). For the groups of mice treated with DLX2323 and canakinumab onlyvery low amounts of mIL-6 or even no mIL-6 could be detected (0.0-2pg/ml of mIL-6; FIG. 4). Mice receiving PBS or control scFv showedsignificantly elevated IL-6 levels of 50 to 170 pg/ml. DLX2323 was veryefficiently neutralizing human IL-1 beta in an in vivo setting, even ata 5 mg/kg dose.

Example 9—CDR Libraries

To better characterize the association of DLX2323 with human IL-1 beta,amino acid mutations were designed and site-specifically inserted intothe CDR regions of DLX2323. CDR positions were chosen for mutagenesisbased on their surface exposure, anticipated interaction with human IL-1beta as deduced from homology models or sequence comparisons withantecedent rabbit IgGs. In the light chain, the motif DNW in CDR-L1 (SEQID No: 14), the amino acid residues R and T in CDR-L2 (SEQ ID No: 15),the threonine in CDR-L3 as well as the motif GGVS were selected (SEQ IDNo: 16) for the design of variants of DLX2323. In the heavy chain, themotif SA was chosen in CDR-H1 (SEQ ID No: 11), in the CDR-H2 the motifYD and the following tyrosine (SEQ ID No: 12). In CDR-H3, all positionswere selected for substitution (SEQ ID No: 13) except for the N-terminalglutamic acid (E). All said positions are indicated with an X in SEQ IDNos. 11-16, respectively. Site directed mutagenesis, sequencing ofclones and library assembly were carried out by GeneArt (LIFETECHNOLOGIES™, Regensburg, Germany).

The resulting scFv mutants are expressed in 96-well format in 1 mlcultures in E. coli BL21 (DE3) (Novagen, USA, cat. no. 69450-3). Duringexpression in this system most of the scFv proteins form insolubleinclusion bodies within the cell but a considerable amount of proteincan be retrieved from the soluble fractions after cell lyses, which wasfound to be sufficient for the analysis by the rhIL-1 beta ELISA (seeexample 2 for details). Cells were lysed in lysis buffer (1 mM EDTA, 0.1mg/ml lysozyme, PBS pH 7.2) in a 96-well format by freezing andsubsequent thawing. Derived crude extracts were cleared bycentrifugation. Supernatants were added to wells of a microtiterplatecoated with 50 ng/ml of rhIL-1 beta (Peprotech) per well. After washing,bound scFvs are detected by ProteinL-HRP and binding to rhIL-1 betaquantified by BM Blue POD Substrate (Roche Applied Science).

Table 3 lists single-site mutations that clearly permit binding ofrhIL-1 beta as defined by an ELISA signal at least 2-fold over thenegative control, but not smaller than 0.1 optical units.

TABLE 3 Residue position amino acid substitutions CDR-L1_D32 A, C, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y CDR-L1_N33 A, C, D, E, F, G,H, I, K, L, M, P, Q, S, T, V, W, Y CDR-L1_W40 E, F, G, M, N, Q, S, YCDR-L2_R58 A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, W, YCDR-L2_T69 A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, YCDR-L3_T109 A, C, I, N, S, V CDR-L3_G111 A, P, S CDR-L3_G112 A, C, D, E,F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y CDR-L3_V135 A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, W, Y CDR-L3_S136 A, C, D, E, F, G,H, I, L, M, N, P, Q, R, T, V, W, Y CDR-H1_S33 A, C, D, E, F, G, H, I, K,L, M, N, P, Q, R, T, V, W, Y CDR-H1_A39 C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, Y CDR-H2_Y59 A, C, G, M CDR-H2_D60 N, P CDR-H2_Y69 A,D, E, G, F, H, I, K, L, M, N, P, S, T, W CDR-H3_R110 A, C, D, E, F, G,H, I, K, L, M, N, Q, S, T, V, W, Y CDR-H3_A111 C, D, F, G, H, I, K, M,N, P, Q, R, S, T, V, W, Y CDR-H3_I112 A, C, F, H, L, M, N, Q, S, T, V, YCDR-H3_F113 I CDR-H3_S114 A, C, E, G, T, V CDR-H3_G115 A, M, N,CDR-H3_D135 A, E, H, N, S, T CDR-H3_F136 A, C, G, H, I, L, M, N, Q, S,T, V, W, Y CDR-H3_V137 A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, W, Y CDR-H3_L138 A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,W, Y

These binding data have predictive value also concerning theneutralization of rhIL-1 beta biological function as verified by theexpression of five exemplary single-site mutants in large scale format,purified to homogeneity, and subjected to cell-based analysis alongsidewith likewise purified DLX2323 protein.

DLX2323_CDR-H1_A39N (SEQ ID No. 45 with X=N), DLX2323_CDR-H3_A111N (SEQID No.: 50 with X=N), DLX2323 CDR-H3_F136N (SEQ ID No. 56 with X=N),DLX2323_CDR-H3_V137D (SEQ ID No.: 57 with X=D), and DLX2323_CDR-H3_L138D(SEQ ID No.: 58 with X=D) and the corresponding parental scFv, DLX2323,were expressed in E. coli BL21(DE3) (Novagen) cells at a 2 liter scale.Following cell lysis by sonication, inclusion bodies were enriched bycentrifugation, washed, protein solubilized in guanidine buffer (6 Mguanidine/HCl, 100 mM Tris, 1 mM EDTA, pH 8.5), refolded in urea buffer(4 M urea, 50 mM glycine, 2 mM cystine, 2 mM cysteine, pH10),concentrated in 50 mM glycine, 50 mM NaCl, pH10, and purified tohomogeneity by size exclusion chromatography. Monomeric peak fractionsof this procedure were concentrated to a final protein content ofapproximately 0.5-5 mg/ml, passed over a sterile filter unit, and assuch subjected to cell-based analyses as described in Example 3. Table 4shows the neutralization potency IC₅₀ of each of the mutants asdetermined in two independent runs.

TABLE 4 Clone ID IC₅₀ DLX2323 2-4 pM DLX2323_CDR-H1_A39N 10 pMDLX2323_CDR-H3_A111N 2-4 pM DLX2323_CDR-H3_F136N 2-4 pMDLX2323_CDR-H3_V137D 5 pM DLX2323_CDR-H3_L138D 10 pM

In overall good agreement with the preceding ELISA results, an IC₅₀value in the low pM range was observed for all five single-site mutantsselected. Each member of the selected mutant subset had a neutralizingcapacity towards rhIL-1 beta comparable to that of the parental scFvDLX2323. Accordingly, the binding of DLX2323 and its variants to rhIL-1beta shows that a significant degree of sequence variability is allowedwhile concomitantly preserving key functional features.

Example 10—Generation of Combinatorial Variants

Based on the above, combinatorial mutants of DLX2323 were designedcombining 3 to 9 of the previously investigated CDR singles-sitechanges. Individual residue changes were selected on basis of: (i)strong ELISA binding results comparable to or better than that of theparent antibody DLX2323; (ii) combinations of far-spread single sitechanges that would simultaneously affect multiple CDR regions; and (iii)a cumulative modulation of CDR-H3, alone. The designed mutants wereexpressed in large scale format and purified as described above for thesingle mutants, before the neutralization capacity was assessed in thehuman fibroblast assay of Example 3. Individual amino acid changes persequence and IC₅₀ data of corresponding scFv proteins are detailed inTable No.: 5. All these isolated scFv proteins had neutralizing activitytowards rhIL1-beta in vitro. The five best candidates displayed IC₅₀values in the low picomolar range, and are thus highly comparable to theparental scFv DLX2323.

TABLE 5 scFv Amino acid changes (AHo annotation) IC50 DLX2464 VL:parental  4 pM VH: A111Q, F136M, V137A, L138G DLX2465 VL: parental  4 pMVH: A111N, F136M, V137D DLX2466 VL: D32G, N33K, W40Y, T109A 300 pM VH:A39D, A111N, F136M, V137A, L138G DLX2467 VL: D32H, T69N, V135S  25 pMVH: S33R, A111M DLX2468 VL: D32G, W40Y, T109A, V135T  15 pM VH: A39S,A111N, F136M DLX2475 VL: N33K, R58Q, S136H 200 pM VH: A39Y, V137KDLX2476 VL: D32S, N33S 100 pM VH: A111D, L138G DLX2480 VL: V135I, S136N 5 pM VH: S33D, A111Q

Variants carrying more than 6 amino acid substitutions in the CDR andflanking framework regions of the variable light and/or the variableheavy chain were designed. In total, 10 combinatorial candidates (fourVL and six VH mutant sequences) were generated, expressed, andfunctionally analysed for rhIL1-beta binding (ELISA) and neutralization(human fibroblast assay) in vitro as described in Example 3. Table 6summarizes the respective point mutations as well as the neutralizationpotency as measured in two independent experiments of each clone.

TABLE 6 ScFv Amino acid changes (AHo annotation) IC50 DLX2543 VL: I20T,D32S, N33S, R58K, A87T, T109A 3-4 pM VH: parental DLX2529 VL: Q24R,D32G, T69N, D99E, V135I, S136N 1-2 pM VH: parental DLX2547 VL: E1D,I20T, D32S, N33S, W40Y, V135I, S136N, T146E, 30 pM V147I, L148K, G149RVH: parental DLX2528 VL: 120T, D32G, S42A, R58Q, T69N, A87T, E88D,T109A, 8 pM G112A, V135T, S136T VH: parental DLX2585 VL: parental 1-2 pMVH: A25V, A39Y, A42S, R82K, V137D, L138Y DLX2545 VL: parental 7 pM VH:V55I, V89L, Y105F, A111N, F136M, L144T DLX2531 VL: parental 0.6-1 pM VH:L12N, S33N, A39S, T84N, V103T, V137A DLX2586 VL: parental 2 pM VH: A25V,A39Y, A42S, V55I, R82K, Y105F, A111Q, F136M, V137D, L138G DLX2530 VL:parental 1-2 pM VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,V137A, L144T DLX2548 VL: parental 1-2 pM VH: A25V, A39Y, A42S, V55I,R82K, V89L, Y105F, A111Q, F136M, V137D, L138G, L144T

All 10 scFv proteins were found to effectively neutralize rhIL1-beta inthe human fibroblast assay in vitro with IC₅₀ values in the highfemtomolar to low picomolar range. Clones DLX2531, DLX2548, DLX2585,DLX2530, DLX2529, and DLX2586 reproducibly yielded lower IC₅₀ valuesthan the parental scFv DLX2323.

Finally, the above VL and VH sequences were chain shuffled. Binding torhIL1-beta was confirmed for cleared lysates of E. coli BL21 origamicells transformed with 19 chain shuffled VL and VH mutants. Table 7summarizes the substitutions.

TABLE 7 VL-VH ScFv fusion Amino acid substitutions DLX2676 DLX2528_ VL:I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A, 2530 G112A, V135T,S136T VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M, V137A,L144T DLX2677 DLX2528_ VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D,T109A, 2531 G112A, V135T, S136T VH: L12N, S33N, A39S, T84N, V103T, V137ADLX2678 DLX2528_ VL: I20T, D32G, S42A, R58Q, T69N, A87T, E88D, T109A,2548 G112A, V135T, S136T VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F,A111Q, F136M, V137D, L138G, L144T DLX2679 DLX2528_ VL: I20T, D32G, S42A,R58Q, T69N, A87T, E88D, T109A, 2585 G112A, V135T, S136T VH: A25V, A39Y,A42S, R82K, V137D, L138Y DLX2680 DLX2529_ VL: Q24R, D32G, T69N, D99E,V135I, S136N 2530 VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M,V137A, L144T DLX2681 DLX2529_ VL: Q24R, D32G, T69N, D99E, V135I, S136N2531 VH: L12N, S33N, A39S, T84N, V103T, V137A DLX2682 DLX2529_ VL: Q24R,D32G, T69N, D99E, V135I, S136N 2548 VH: A25V, A39Y, A42S, V55I, R82K,V89L, Y105F, A111Q, F136M, V137D, L138G, L144T DLX2683 DLX2529_ VL:Q24R, D32G, T69N, D99E, V135I, S136N 2585 VH: A25V, A39Y, A42S, R82K,V137D, L138Y DLX2684 DLX2543_ VL: I20T, D32S, N33S, R58K, A87T, T109A2530 VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N, F136M, V137A, L144TDLX2685 DLX2543_ VL: I20T, D32S, N33S, R58K, A87T, T109A 2531 VH: L12N,S33N, A39S, T84N, V103T, V137A DLX2686 DLX2543_ VL: I20T, D32S, N33S,R58K, A87T, T109A 2548 VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F,A111Q, F136M, V137D, L138G, L144T DLX2687 DLX2543_ VL: I20T, D32S, N33S,R58K, A87T, T109A 2585 VH: A25V, A39Y, A42S, R82K, V137D, L138Y DLX2689DLX2544_ VL: E1D, I20T, D32S, N33S, W40Y, R58K, A87T, E88D, D99E, 2531T109A, V135I, S136N, L145V, T146E, V147I, L148K, G149R VH: L12N, S33N,A39S, T84N, V103T, V137A DLX2690 DLX2544_ VL: E1D, I20T, D32S, N33S,W40Y, R58K, A87T, E88D, D99E, 2548 T109A, V135I, S136N, L145V, T146E,V147I, L148K, G149R VH: A25V, A39Y, A42S, V55I, R82K, V89L, Y105F,A111Q, F136M, V137D, L138G, L144T DLX2691 DLX2544_ VL: E1D, I20T, D32S,N33S, W40Y, R58K, A87T, E88D, D99E, 2585 T109A, V135I, S136N, L145V,T146E, V147I, L148K, G149R VH: A25V, A39Y, A42S, R82K, V137D, L138YDLX2692 DLX2547_ VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E,2530 V147I, L148K, G149R VH: L12N, S33D, Y69F, T84N, V89L, V103T, A111N,F136M, V137A, L144T DLX2693 DLX2547_ VL: E1D, I20T, D32S, N33S, W40Y,V135I, S136N, T146E, 2531 V147I, L148K, G149R VH: L12N, S33N, A39S,T84N, V103T, V137A DLX2694 DLX2547_ VL: E1D, I20T, D32S, N33S, W40Y,V135I, S136N, T146E, 2548 V147I, L148K, G149R VH: A25V, A39Y, A42S,V55I, R82K, V89L, Y105F, A111Q, F136M, V137D, L138G, L144T DLX2695DLX2547_ VL: E1D, I20T, D32S, N33S, W40Y, V135I, S136N, T146E, 2585V147I, L148K, G149R VH: A25V, A39Y, A42S, R82K, V137D, L138Y

Binding was considered specific if an ELISA signal of >0.1 optical unitswas obtained which was at least 2-fold higher than the negative control.Results are shown in FIG. 6. For example, DLX2690 and DLX2691 slightlypassed the cut-off filter, but are tested positive. The remainingcollective VL and VH chain-shuffled mutants bind rhIL1-beta withabsolute ELISA signals comparable to or even higher than DLX2323.

The IC₅₀ values of DLX2681 and DLX2693 were determined in the humanfibroblast assay as described above. DLX2681 was found to neutralizerhIL1-beta with an IC₅₀ value of 0.6 pM and the IC₅₀ value of DLX2693was determined to be 12.5 pM.

While there are shown and described presently preferred embodiments ofthe invention, it is to be understood that the invention is not limitedthereto but may be otherwise variously embodied and practiced within thescope of the following claims. Since numerous modifications andalternative embodiments of the present invention will be readilyapparent to those skilled in the art, this description is to beconstrued as illustrative only and is for the purpose of teaching thoseskilled in the art the best mode for carrying out the present invention.Accordingly, all suitable modifications and equivalents may beconsidered to fall within the scope of the following claims.

1-29. (canceled)
 30. A method of producing an antibody or a fragmentthereof against IL-1 beta, wherein the antibody or a fragment thereofcomprises: a. the variable heavy chain (VH) CDR sequences CDR-H1,CDR-1H2 or CDR-H3 as set forth in: SEQ ID NOS: 1, 2 and 3, respectively;and b. the variable light chain (VL) CDR sequences CDR-L1, CDR-L2 orCDR-L3 as set forth in: SEQ ID NOS: 4, 5, and 6, respectively;comprising the steps of: (i) cultivating a host cell comprising anucleic acid encoding the antibody or a fragment thereof so that theantibody or a fragment thereof is expressed; (ii) recovering; and (iii)purifying the antibody or a fragment thereof.
 31. The method of claim30, wherein the antibody or a fragment thereof has a potency (IC₅₀) withregard to inhibiting the biological effect of human IL-1 beta of lowerthan 50 pM as determined by inhibiting IL-1 beta stimulated release ofIL-6 from human fibroblasts.
 32. The method of claim 30, wherein theantibody or a fragment thereof is a Fab, a Fab′, a scFv, or a Fvfragment.
 33. The method of claim 30, wherein the antibody or a fragmentthereof is a full-length immunoglobulin or a bivalent antibody fragment.34. The method of claim 30, wherein the antibody or a fragment thereofcomprises the light chain variable framework region FR-L1 of SEQ ID NO:18, the light chain variable framework region FR-L2 of SEQ ID NO: 19,the light chain variable framework region FR-L3 of SEQ ID NO: 20 and/orthe light chain variable framework region FR-L4 of SEQ ID NO:
 21. 35.The method of claim 30, wherein the antibody or a fragment thereofcomprises the heavy chain variable framework region FR-H1 of SEQ ID NO22; the heavy chain variable framework region FR-H2 of SEQ ID NO: 23;the heavy chain variable framework region FR-H3 of SEQ ID NO: 24; and/orthe heavy chain variable framework region FR-H4 of SEQ ID NO:
 25. 36.The method of claim 30, wherein the antibody or a fragment thereofcomprises: a. a VH sequence of SEQ ID NO: 7; and b. a VL sequence of SEQID NO:
 8. 37. The method of claim 30, wherein the antibody or a fragmentthereof comprises the linker sequence of SEQ ID NO:
 9. 38. The method ofclaim 30, wherein the antibody or a fragment thereof has the sequence ofSEQ ID NO:
 10. 39. The method of claim 30, wherein the antibody or afragment thereof is humanized.
 40. A method of producing an antibody ora fragment thereof against IL-1 beta, wherein the antibody or a fragmentthereof comprises: a. the variable heavy chain (VH) CDR sequencesCDR-H1, CDR-H2 or CDR-H3 as set forth in: SEQ ID NOS: 1, 2 and 3,respectively; and b. the variable light chain (VL) CDR sequences CDR-L1,CDR-L2 or CDR-L3 as set forth in: SEQ ID NOS: 4, 5, and 6, respectively;comprising the steps of: (i) providing a cell-free system; (ii)providing a nucleic acid encoding the antibody or a fragment; (iii)allowing for transcription and translation of said nucleic acid producttemplate; (iv) recovering; and (v) optionally purifying the antibody ora fragment thereof.
 41. The method of claim 40, wherein the antibody ora fragment thereof has a potency (IC₅₀) with regard to inhibiting thebiological effect of human IL-1 beta of lower than 50 pM as determinedby inhibiting IL-1 beta stimulated release of IL-6 from humanfibroblasts.
 42. The method of claim 40, wherein the antibody or afragment thereof is a Fab, a Fab′, a scFv, or a Fv fragment.
 43. Themethod of claim 40, wherein the antibody or a fragment thereof is afull-length immunoglobulin or a bivalent antibody fragment.
 44. Themethod of claim 40, wherein the antibody or a fragment thereof comprisesthe light chain variable framework region FR-L1 of SEQ ID NO: 18, thelight chain variable framework region FR-L2 of SEQ ID NO: 19, the lightchain variable framework region FR-L3 of SEQ ID NO: 20 and/or the lightchain variable framework region FR-L4 of SEQ ID NO:
 21. 45. The methodof claim 40, wherein the antibody or a fragment thereof comprises theheavy chain variable framework region FR-H1 of SEQ ID NO 22; the heavychain variable framework region FR-H2 of SEQ ID NO: 23; the heavy chainvariable framework region FR-H3 of SEQ ID NO: 24; and/or the heavy chainvariable framework region FR-H4 of SEQ ID NO:
 25. 46. The method ofclaim 40, wherein the antibody or a fragment thereof comprises: a. a VHsequence of SEQ ID NO: 7; and b. a VL sequence of SEQ ID NO:
 8. 47. Themethod of claim 40, wherein the antibody or a fragment thereof comprisesthe linker sequence of SEQ ID NO:
 9. 48. The method of claim 40, whereinthe antibody or a fragment thereof has the sequence of SEQ ID NO: 10.49. The method of claim 40, wherein the antibody or a fragment thereofis humanized.
 50. A method of producing an antibody or a fragmentthereof against IL-1 beta, wherein the antibody or a fragment thereofcomprises: a. the variable heavy chain (VH) CDR sequences CDR-H1, CDR-H2or CDR-1H3 as set forth in: SEQ ID NOS: 1, 2 and 3, respectively; and b.the variable light chain (VL) CDR sequences CDR-L1, CDR-L2 or CDR-L3 asset forth in: SEQ 1D NOS: 4, 5, and 6, respectively; comprising at leastone step of chemical synthesis.